Extensive apoptosis during the formation of the terminal nerve ganglion by olfactory placode-derived cells with distinct molecular markers.

The terminal nerve ganglion (TNG) is a well-known structure of the peripheral nervous system in cartilaginous and teleost fishes. It derives from the olfactory placode during embryonic development. While the differentiation and migration of gonadotropin releasing hormone (GnRH)-expressing neurons from the olfactory placode has been well documented, the TNG has been neglected in birds and mammals, and its development is less well described. Here we describe the formation of a ganglion-like structure from migratory olfactory placodal cells in chicken. The TNG is surrounded by neural crest cells, but in contrast to other cranial sensory ganglia, we observed no neural crest corridor, and olfactory unsheathing cells appear only after the onset of neuronal migration. We identified Isl1 and Lhx2 as two transcription factors that label neuronal subpopulations in the forming TNG, distinct from GnRH1+ cells, thereby revealing a diversity of cell types during the formation of the TNG. We also provide evidence for extensive apoptosis in the terminal nerve ganglion shortly after its formation, but not in other cranial sensory ganglia. Moreover, at later stages placode-derived neurons expressing GnRH1, Isl1 and/or Lhx2 become incorporated in the telencephalon. The integration of TNG neurons into the telencephalon together with the earlier widespread apoptosis in the TNG might be an explanation why the TNG in mammals and birds is much smaller compared to other vertebrates.

Preclinical Study in Mouse Thymus and Thymocytes: Effects of Treatment with a Combination of Sodium Dichloroacetate and Sodium Valproate on Infectious Inflammation Pathways.

The research presents data from a preclinical study on the anti-inflammatory effects of a sodium dichloroacetate and sodium valproate combination (DCA-VPA). The 2-week treatment with a DCA 100 mg/kg/day and VPA 150 mg/kg/day combination solution in drinking water's effects on the thymus weight, its cortex/medulla ratio, Hassall's corpuscles (HCs) number in the thymus medulla, and the expression of inflammatory and immune-response-related genes in thymocytes of male Balb/c mice were studied. Two groups of mice aged 6-7 weeks were investigated: a control (n = 12) and a DCA-VPA-treated group (n = 12). The treatment did not affect the body weight gain (p > 0.05), the thymus weight (p > 0.05), the cortical/medulla ratio (p > 0.05), or the number of HCs (p > 0.05). Treatment significantly increased the Slc5a8 gene expression by 2.1-fold (p < 0.05). Gene sequence analysis revealed a significant effect on the expression of inflammation-related genes in thymocytes by significantly altering the expression of several genes related to the cytokine activity pathway, the inflammatory response pathway, and the Il17 signaling pathway in thymocytes. Data suggest that DCA-VPA exerts an anti-inflammatory effect by inhibiting the inflammatory mechanisms in the mouse thymocytes.

CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells.

The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form secondary tumors). Delamination, defined as the degradation of the basal lamina and basement membrane, is the initial process that facilitates the transmigration and spread of cancer cells to other tissues and organs. Scoring the delamination capacity of cancer cells would indicate the metastatic potential of these cells. We have developed a standardized method, the ex ovo CAM-Delam assay, to visualize and quantify the ability of cancer cells to delaminate and invade, thereby being able to assess metastatic aggressiveness. Briefly, the CAM-Delam method includes seeding cancer cells in silicone rings on the chick chorioallantoic membrane (CAM) at embryonic day 10, followed by incubation from hours to a few days. The CAM-Delam assay includes the use of an internal humidified chamber during chick embryo incubation. This novel approach increased embryo survival from 10%-50% to 80%-90%, which resolved previous technical problems with low embryo survival rates in different CAM assays. Next, the CAM samples with associated cancer cell clusters were isolated, fixed, and frozen. Finally, cryostat-sectioned samples were visualized and analyzed for basement membrane damage and cancer cell invasion using immunohistochemistry. By evaluating various known metastatic and non-metastatic cancer cell lines designed to express green fluorescent protein (GFP), the CAM-Delam quantitative results showed that the delamination capacity patterns reflect metastatic aggressiveness and can be scored into four categories. Future use of this assay, apart from quantifying delamination capacity as an indication of metastatic aggressiveness, is to unravel the molecular mechanisms that control delamination, invasion, the formation of micrometastases, and changes in the tumor microenvironment.

Tumors derived from lung cancer cells respond differently to treatment with sodium valproate (a HDAC inhibitor) in a chicken embryo chorioallantoic membrane model.

Lung cancer is the most frequent cause of cancer death. Some human lung malignant tumors have a combined small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) histology, with tumor cell phenotype changing during tumor progression. Valproic acid is used as an anti-seizure medication to treat migraine, and bipolar mood disorders. Recently, its efficacy as an adjuvant therapy was shown in cancer due to its histone deacetylase (HDAC) inhibitory property. HDACs are upregulated in lung tumors, and HDAC inhibitors, including valproic acid, inhibit endothelial cell proliferation in vitro and in vivo and have antiproliferative and antimigratory properties. We tested valproic acid for possible antiangiogenic and antimigratory effects on experimental lung tumors grafted onto the chicken embryo chorioallantoic membrane (CAM). Tumors were formed from two NSCLC cell lines and a single SCLC cell line. To investigate tumor and CAM interactions, in vivo biomicroscopy, visualization of blood vessels with injected fluorescent dextran, histological, immunohistochemical and histomorphometric methods were applied. Our results showed that a sodium valproate (NaVP) treatment-induced a dose-dependent decrease of experimental tumor invasion into the CAM mesenchyme and a reduction in angiogenesis. Both the invasion and the angiogenic response were dependent on the type of cell line used: invasion and angiogenesis of tumors derived from A549 and NCI-H146 cell lines responded to increasing doses of NaVP from 4 to 8 mM, whereas Sk_Lu_1 cells response were antimigratory and antiangiogenic when NaVP was used up to 6 mM. When 8mM NaVP was used, stimulated invasion and angiogenesis in tumors from Sk_Lu_1 cells were observed.

CAM-Delam: an in vivo approach to visualize and quantify the delamination and invasion capacity of human cancer cells.

The development of metastases is the major cause of cancer related death. To develop a standardized method that define the ability of human cancer cells to degrade the basement membrane, e.g. the delamination capacity, is of importance to assess metastatic aggressiveness. We now present the in vivo CAM-Delam assay to visualize and quantify the ability of human cancer cells to delaminate and invade. The method includes seeding cancer cells on the chick chorioallantoic membrane (CAM), followed by the evaluation of cancer-induced delamination and potential invasion within hours to a few days. By testing a range of human cancer cell lines in the CAM-Delam assay, our results show that the delamination capacity can be divided into four categories and used to quantify metastatic aggressiveness. Our results emphasize the usefulness of this assay for quantifying delamination capacity as a measurement of metastatic aggressiveness, and in unraveling the molecular mechanisms that regulate delamination, invasion, formation of micro-metastases and modulations of the tumor microenvironment. This method will be useful in both the preclinical and clinical characterization of tumor biopsies, and in the validation of compounds that may improve survival in metastatic cancer.

Sodium Valproate Inhibits Small Cell Lung Cancer Tumor Growth on the Chicken Embryo Chorioallantoic Membrane and Reduces the p53 and EZH2 Expression.

The study aims to test the effect of different sodium valproate (NaVP) doses on small cell lung cancer NCI-H146 cells tumor in chicken embryo chorioallantoic membrane (CAM) model. Xenografts were investigated in the following groups: nontreated control and 5 groups treated with different NaVP doses (2, 3, 4, 6, and 8 mmol/L). Invasion of tumors into CAM in the nontreated group reached 76%. Tumors treated with 8 mmol/L NaVP doses significantly differed in tumor invasion frequency from the control and those treated with 2 mmol/L (P < .01). The calculated probability of 50% tumor noninvasion into CAM was when tumors were treated with 4 mmol/L of NaVP. Number of p53-positive cells in tumors was significantly reduced when treated with NaVP doses from 3 to 8 mmol/L as compared with control; number of EZH2-positive cells in control significantly differed from all NaVP-treated groups. No differences in p53- and EZH2-positive cell numbers were found among 4, 6, and 8 mmol/L NaVP-treated groups. Invaded tumors had an increased N-cadherin and reduced E-cadherin expression. The results indicate the increasing NaVP dose to be able to inhibit tumors progression. Expression of p53 and EZH2 may be promising target markers of therapeutic efficacy evaluation.

The Effect of Sodium Valproate on the Glioblastoma U87 Cell Line Tumor Development on the Chicken Embryo Chorioallantoic Membrane and on EZH2 and p53 Expression.

Literature data support evidences that glioblastoma (GBM) patients experience prolonged survival due to sodium valproate (NaVP) treatment. The study assessed the human GBM cell U87 xenograft studied in the chicken embryo chorioallantoic membrane (CAM) model evaluating NaVP effect on tumor. Three groups of tumors (each n = 10) were studied: nontreated, treated with 4 mM, and treated with 8 mM of NaVP. The majority of tumors without NaVP treatment during tumor growth destroyed the chorionic epithelium, invaded the mesenchyme, and induced angiogenesis. Incidence of tumor formation on CAM without invasion into the mesenchyme was higher when U87 cells were treated with NaVP; the effect significantly increased with NaVP concentration. Treatment with 8 mM of NaVP did not show clear dynamics of tumor growth during 5 days; at the same time, the angiogenesis failed. With a strong staining of EZH2, p53 in tumors without NaVP treatment was found, and NaVP significantly decreased the expression of EZH2- and p53-positive cells; the effect was significantly higher at its 8 mM concentration. NaVP has a function in blocking the growth, invasion, and angiogenesis of tumor in the CAM model; tumor growth interferes with EZH2 and p53 molecular pathways, supporting the NaVP potential in GBM therapy.

Sex-related differences of urethane and sodium valproate effects on Ki-67 expression in urethane-induced lung tumors of mice.

The aim of the present study was to evaluate sex differences in tumorigenesis by assessing the number of Ki-67-positive cells [Ki-67(+)] in urethane-induced mice lung tumors and the effect of sodium valproate (NaVP) in BALB/c mice. Gonad-intact and gonadectomized female and male mice were divided into the following groups: i) Treated with urethane, ii) treated with urethane and NaVP and iii) gonad-intact or gonadectomized control. Urethane (total 50 mg/mouse) was injected intraperitoneally. The NaVP 0.4% solution was administered orally for 6 months. Histologically, lung tumors were divided into adenomas and adenocarcinomas and assessed immunohistochemically using antibodies against Ki-67. The Ki-67(+) was calculated per one mm2 of a tumor. In adenomas, Ki-67(+) in the urethane-treated gonad-intact males was significantly higher than in females (P=0.001) and in castrated males (P<0.01); Ki-67(+) in adenomas of the urethane-treated gonad-intact males was significantly higher than in urethane-NaVP-treated ones (P<0.04). No significant differences were found in analogous female groups. In adenocarcinomas, Ki-67(+) in urethane-treated gonad-intact males was significantly higher than in females and gonadectomized mice of both sexes (P<0.001), and in ovariectomized females was significantly higher than in ovary-intact group (P=0.01). A significantly higher number of Ki-67(+) cells were observed in gonad-intact adenocarcinomas of the urethane-NaVP-treated females compared with the urethane-treated ones (P<0.001). Comparing between urethane-NaVP-treated gonadectomized males and females in adenocarcinomas, determined that Ki-67(+) was significantly lower in females (P=0.005). In adenocarcinomas, Ki-67(+) in urethane-NaVP-treated gonadectomized males and females was significantly lower than in gonad-intact mice of the same sex (P<0.001). In summary, gonadectomy with NaVP treatment decreased Ki-67(+) in adenocarcinomas for mice of both sexes. The results of the present study indicate sex-related differences in mice lung tumorigenesis, and a sex-related effect of NaVP on progression in urethane-induced BALB/c mice lung tumors.