Studies Of Expression And Roles Of Slit/Robo And AhR In Human Ovarian Cancer Tissue And Cell Lines

PART I Studies of expression and roles of Slit/Robo in human ovarian cancer tissue and cell linesBackgroundThe secreted Slit glycoproteins and their Roundabout (Robo) receptors, consisting of three Slit (Slitl-3) and four Robo (Robo 1-4) members in vertebrates, were originally identified in the nervous system as a repulsive cue to prevent axons from migrating to inappropriate locations during the assembly of the nervous system. Each mammalian Slit contains four leucine-rich regions (Robo binding domains), nine epidermal growth factor repeats, a laminin G domain, and a C-terminal cysteine-rich domain. Slits can be proteolytically cleaved into the N- and C-terminal fragments in vivo and in vitro. It is believed that only full length and the N-terminus, both of which are predominantly membrane-bound, of the Slits can bind and activate Robos. Robos belong to the Ig superfamily of transmembrane signaling molecules. Robo 1-3 consists of five Ig-like and three type III fibronectin domains in the ectodomain, whereas Robo4 has only two Ig domains. To date, the specific high-affinity Slit binding has been demonstrated biochemically for all Robos, expect for Robo4.The Slit/Robo is expressed in many types of human cancers. Nonetheless, reports on roles of the Slit/Robo in human cancers are controversial. It has been postulated that the Slit/Robo may function as a tumor suppressor. This is supported by several lines of evidence. First, homozygous deletion of ROBO1 gene is detected in lung and breast carcinomas. Second, Slit2 is able to suppress growth of breast and colorectal tumor cells and the Slit/Robo signal is often inactivated in breast, lung, colorectal, and brain cancers by hypermethylation of Slit and Robo promoters. Third, expression of certain Slit/Robo members is decreased in many cancer tissues including lung cancer, breast cancer, adult renal cell carcinoma, cervical cancer and prostate cancer as compared with normal tissues or less advanced cancer tissues. In contrast, increased expression of certain Slit/Robo members has also been reported in prostate cancer, breast cancer, colorectal cancer, hepatocellular carcinoma, lung, kidney, liver, and metastatic melanoma. Furthermore, a recent report has shown that neutralization of Robo 1 reduces the cancer mass and the microvascular density of human malignant melanoma in vivo, suggesting that the Slit/Robo may promote cancer growth and development, plausibly via promoting cancer angiogenesis. Thus, the Slit/Robo might either promote or inhibit cancer development, depending on individual type of cancers.The roles of Slit/Robo have been detected in many human cancer, and according to the recent research, SLIT1,SLIT2,SLIT3 and ROBO1 have emerged as candidate tumour suppressor genes in a variety of epithelial tumor types. As we know,90% of ovarian cancer is from the epithelial cell, but little is known about expression and roles of the Slit/Robo in human ovarian cancer. Herein, for the first time using human ovarian tissue microarray, we examined expression of Slit2/3 and Robo1/4, four major members of the Slit/Robo in human normal and malignant ovary tissues, as well as OVCAR-3 and SKOV-3 cells, two human ovarian cancer cell lines. We also examined potential participation of the Slit/Robo signal in regulating migration and proliferation of SKOV-3 and OVCAR-3 cells.Objective:1. To examine expression of Slit2/3 and Robo1/4, four major members of the Slit/Robo signaling pathway in human normal and malignant ovary tissue; as well as the human ovarian cancer cell; 2. To examine potential participation of the Slit/Robo signaling pathway in regulating migration and proliferation of OVCAR-3 and SKOV-3 cells.3. To examine effects of Slit2 on the levels of phosphor-ERK1/2 and AKT1 in the OVCAR-3 and SKOV-3 cell.Methods:1. Immonohistochemistry:for detect the immunolocalization of Slit2/3 and Robol/4 in the human ovarian cancer tissue microarray.Preimmune goat or rabbit IgG replace primary antibody as the negative control. Staining intensity of Slit2/3 and Robol/4 was semi-quantitatively analyzed.The images were uniformly converted to 8-bit grey scale pictures. The integrated optical density (OD) value was measured by the MetaMorph analysis software.2. Western blot analysis:for expression of Slit2/3 and Robol/4 in OVCAR-3 and SKOV-3 cells.3. Crystal violet method:to detect effects of Slit2 on ovarian cancer cell proliferation.4.24-Multiwell BD Falcon FluoroBlok Insert System:to detect effects of Slit2 on ovarian cancer cell migration.5. Wound healing assay:to confirm Slit2's action on ovarian cancer cell migration and proliferation.6. Western blot analysis:to detect the effects of Slit2 on levels of phosphor-ERK1/2 and AKT1 in OVCAR-3 and SKOV-3 cells.Results:1. Expression of Slit2/3 and Robol/4:Since no significant difference in Slit2/3 and Robol/4 staining intensity was observed between adjacent normal ovarian tissues (n=8) and normal ovarian tissues (n=8), data from these two types of tissues were pooled. Positive staining of Slit2/3 and Robo1, but not Robo4 was observed in human normal ovary tissue, primary in the stromal cell;the immunoreactivity of Slit2/3 and Robol/4 was also found in cancer cells in several different histotypes of ovarian cancer tissues (n=92). No staining in the negative control.1) Slit2:The overall staining intensity was relatively high in NORM, AGCT, DISG, YST, Mu-ADNE, L-Se-ADEN, and H-Se-ADEN as compared with that in ADEN and TMC. The semi-quantitative analysis confirmed these observations. Significant (p≤0.05) differences in the staining intensity was detected only in YST and L-Se-ADEN as compared with NORM;2) Slit3:The strong staining was seen in DISG and YST, while weak staining was presented in NORM, ADEN, AGCT, Mu-ADEN, TMC, L-Se-ADEN, and H-Se-ADEN. The semi-quantitative analysis also indicated that the staining was much more (p≤0.05) intensive in DISG and YST than the others;3) Robol:moderate and strong staining was observed in NORM and all histotypes of ovarian cancer. The semi-quantification analysis revealed that the staining intensity in DISG, YST, Mu-ADEN,L-Se-ADEN, and H-Se-ADEN was significantly higher (p≤0.05) than that in NORM, AGCT, ADEN, and TMC;4) Robo4:the overall staining was weak in NORM and all histotypes of ovarian cancer tissues except DISG, in which the staining was more intensive than that in all other tissues. These observations were confirmed by the semi-quantitative analysis.2. We detected the protein expression of Slit2/3 and Robol/4 in OVCAR-3 and SKOV-3 cells:the Slit2 antibody against the C-terminus of human Slit2 detected a band at～50 kDa, and human hepatoblastoma cells (the positive controls);the Slit3 antibody against the N-terminus of human Slit3 detected two strong bands at～65 and 45 kDa as well as a weak band at-100 kDa as well as in the positive control (mouse thyroid extract);the Robol/4 antibodies detected a single band at～250 and 170 kDa, both of which were full length according to the positive control (HUVE cell).3. Recombinant human Slit2 did not significantly affect proliferation and migration of OVCAR-3 and SKOV-3 cell.Conclusions:1. The staining level of Slit/Robo in ovarian cancer is much higher than that in normal ovary tissue, which indicated Slit/Robo may play important role in the ovarian cancer;2. Slit/Robo signaling may not play an important role in regulating human ovarian cancer cell proliferation and migration. 3. Slit/Robo signaling didn't induce the phosphorylation of ERK1/2 and AKT1 in OVCAR-3 and SKOV-3 cells.PART II Studies of expression of AhR in human ovarian cancer and the effects on proliferation and migration of cellsBackgroundThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor, which transduces extracellular signals through DNA-binding-dependent and-independent mechanisms. The typical AhR ligands, either synthetic or naturally occurring, are halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, among which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and is a classic AhR ligand. Upon binding to its ligands, the AhR translocates to the nuclei, in which it heterodimerizes with AhR nuclear translocator (ARNT), binds to specific enhancer sequences termed dioxin responsive elements (DRE), and activates expression of downstream genes including CYP1A1, CYP1A2, CYP1B1 (three well studied xenobiotic metabolizing enzymes), p53, and p21.2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous AhR ligand (although arguable), was first isolated from porcine lung tissues. Based on its activity on the DRE-containing reporter gene, the biological potency of ITE was 5-fold greater than that ofβ-nephthoflavone (BNF, one artificial AhR ligand), but～100-fold lower than that of TCDD. Moreover, the estimated Kd value for ITE in mouse hepatoma cells was～5-6 fold greater that of TCDD. However, given that its naturally producing feature and specific binding to the AhR, ITE could be potentially used as an anti-cancer drug, particularly since its lack of toxicity (cleft palate and hydornephrosis) typically associated with TCDD exposure has been reported in the mouse fetus.Roles of the AhR in regulating human ovarian cancer growth remains unknown, although the AhR is known to be expressed in human ovarian cancer and TCDD can stimulate proliferation of CAOV-3 cells, a human ovarian cancer cell line. Herein, we examined expression and roles of the AhR in regulating growth of human ovarian cancer using ex vivo, in vivo and in vitro models.Objective:1. To examine expression of aryl hydrocarbon receptor (AhR) in human normal and malignant ovary tissue.2. To examine the effects of 1TE in regulating migration and proliferation of SKOV-3 and OVCAR-3 cells.Methods:1. Immunohistochemistry:for detect the immunolocalization of AhR in the human ovarian cancer tissue microarray.Preimmune goat or rabbit IgG replace primary antibody as the negative control. Staining intensity of AhR was semi-quantitatively analyzed.The images were uniformly converted to 8-bit grey scale pictures. The integrated optical density (OD) value was measured by the MetaMorph analysis software.2. Crystal violet mothod:to detect effects of ITE on ovarian cancer cell proliferation.3.24-Multiwell BD Falcon FluoroBlok Insert System:to detect effects of ITE on ovarian cancer cell migration.Results:1. Expression of AhR:the AhR immunoreactivity was present in DISG, ADEN, TMC, YST, Mu-ADEN, and L- and H-Se-ADEN, but not in NORM and AGCT. In all histotypes of adenocarcinomas, the AhR staining was present primarily in cytoplasm of epithelium, but not in other cell types. No positive staining was observed in the preimmune rabbit IgG control (data not shown). The semi-quantification analysis revealed that the OD value in NORM was similar to that in DISG,and ADEN, but was much lower (p≤0.05) than that in TMC, YST, Mu-ADEN, and L- and H-Se-ADEN, in which no difference was detected between the grades, stages, and TNM classifications for each histotype of tumor.2. Treating cells with ITE at any doses studied for two days did not affect OVCAR-3 cell proliferation. ITE at 0.1 nM decreased (p≤0.05) OVCAR-3 cell numbers by 27% and 40% on Days 4 and 6, respectively. ITE from 1 nM to 5μM similarly inhibited (p≤0.01) OVCAR-3 cell numbers by 50% and 66% on Days 4 and 6, respectively3. Treating cells with ITE, but not TCDD for 2,4, and 6 days similarly inhibited (p≤0.05) SKOV-3 cell migration by～54%Conclusions:1. There is no staining of AhR in normal ovary tissue, and strong staining in ovarian cancer tissue, which indicated AhR may play important role in the ovarian cancer;2. ITE inhibited the proliferation of OVCAR-3 cell by activating AhR.3. ITE inhibited the migration of SKOV-3 cell by activating AhR.