The Comparative Experimental Study of Sodium and Magnesium Dichloroacetate Effects on Pediatric PBT24 and SF8628 Cell Glioblastoma Tumors Using a Chicken Embryo Chorioallantoic Membrane Model and on Cells In Vitro.

In this study, pyruvate dehydrogenase kinase-1 inhibition with dichloroacetate (DCA) was explored as an alternative cancer therapy. The study's aim was to compare the effectiveness of NaDCA and MgDCA on pediatric glioblastoma PBT24 and SF8628 tumors and cells. The treatment effects were evaluated on xenografts growth on a chicken embryo chorioallantoic membrane. The PCNA, EZH2, p53, survivin expression in tumor, and the SLC12A2, SLC12A5, SLC5A8, CDH1, and CDH2 expression in cells were studied. The tumor groups were: control, cells treated with 10 mM and 5 mM of NaDCA, and 5 mM and 2.5 mM of MgDCA. The cells were also treated with 3 mM DCA. Both the 10 mM DCA preparations significantly reduced PBT24 and SF8624 tumor invasion rates, while 5 mM NaDCA reduced it only in the SF8628 tumors. The 5 mM MgDCA inhibited tumor-associated neoangiogenesis in PBT24; both doses of NaDCA inhibited tumor-associated neoangiogenesis in SF8628. The 10 mM DCA inhibited the expression of markers tested in PBT24 and SF8628 tumors, but the 5 mM DCA affect on their expression depended on the cation. The DCA treatment did not affect the SLC12A2, SLC12A5, and SLC5A8 expression in cells but increased CDH1 expression in SF8628. The tumor response to DCA at different doses indicated that a contrast between NaDCA and MgDCA effectiveness reflects the differences in the tested cells' biologies.

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.

SARS-CoV-2 Infection, Sex-Related Differences, and a Possible Personalized Treatment Approach with Valproic Acid: A Review.

Sex differences identified in the COVID-19 pandemic are necessary to study. It is essential to investigate the efficacy of the drugs in clinical trials for the treatment of COVID-19, and to analyse the sex-related beneficial and adverse effects. The histone deacetylase inhibitor valproic acid (VPA) is a potential drug that could be adapted to prevent the progression and complications of SARS-CoV-2 infection. VPA has a history of research in the treatment of various viral infections. This article reviews the preclinical data, showing that the pharmacological impact of VPA may apply to COVID-19 pathogenetic mechanisms. VPA inhibits SARS-CoV-2 virus entry, suppresses the pro-inflammatory immune cell and cytokine response to infection, and reduces inflammatory tissue and organ damage by mechanisms that may appear to be sex-related. The antithrombotic, antiplatelet, anti-inflammatory, immunomodulatory, glucose- and testosterone-lowering in blood serum effects of VPA suggest that the drug could be promising for therapy of COVID-19. Sex-related differences in the efficacy of VPA treatment may be significant in developing a personalised treatment strategy for COVID-19.

Different Effects of Valproic Acid on SLC12A2, SLC12A5 and SLC5A8 Gene Expression in Pediatric Glioblastoma Cells as an Approach to Personalised Therapy.

Valproic acid (VPA) is a histone deacetylase inhibitor with sex-specific immunomodulatory and anticancer effects. This study aimed to investigate the effect of 0.5 and 0.75 mM VPA on NKCC1 (SLC12A2), KCC2 (SLC12A5) and SLC5A8 (SLC5A8) co-transporter gene expressions in pediatric PBT24 (boy's) and SF8628 (girl's) glioblastoma cells. The SLC12A2, SLC12A5 and SLC5A8 RNA expressions were determined by the RT-PCR method. The SLC12A2 and SLC5A8 expressions did not differ between the PBT24 and SF8628 controls. The SLC12A5 expression in the PBT24 control was significantly higher than in the SF8628 control. VPA treatment significantly increased the expression of SLC12A2 in PBT24 but did not affect SF8628 cells. VPA increased the SLC12A5 expression in PBT24 and SF8628 cells. The SLC12A5 expression of the PBT24-treated cells was significantly higher than in corresponding SF8628 groups. Both VPA doses increased the SLC5A8 expression in PBT24 and SF8628 cells, but the expression was significantly higher in the PBT24-treated, compared to the respective SF8628 groups. The SLC5A8 expression in PBT24-treated cells was 10-fold higher than in SF8628 cells. The distinct effects of VPA on the expression of SLC12A2, SLC12A5 and SLC5A8 in PBT24 and SF8628 glioblastoma cells suggest differences in tumor cell biology that may be gender-related.

The Different Temozolomide Effects on Tumorigenesis Mechanisms of Pediatric Glioblastoma PBT24 and SF8628 Cell Tumor in CAM Model and on Cells In Vitro.

It is necessary to elucidate the individual effects of temozolomide (TMZ) on carcinogenesis and tumor resistance to chemotherapy mechanisms. The study aimed to investigate the TMZ 50 and 100 µM dose effect difference between PBT24 and SF8628 cell line high-grade pediatric glioblastoma (phGBM) xenografts in a chicken chorioallantoic membrane (CAM) model, on PCNA and EZH2 immunohistochemical expression in the tumor and on the expression of NKCC1, KCC2, E- and N-cadherin genes in TMZ-treated and control cell groups in vitro. TMZ at a 100 µg dose reduced the incidence of PBT24 xenograft invasion into the CAM, CAM thickening and the number of blood vessels in the CAM (p < 0.05), but did not affect the SF8628 tumor in the CAM model. The TMZ impact on PBT24 and SF8628 tumor PCNA expression was similarly significantly effective but did not alter EZH2 expression in the studied tumors. The TMZ at 50 µM caused significantly increased RNA expression of the NKCC1 gene in both studied cell types compared with controls (p < 0.05). The expression of the KCC2 gene was increased in PBT24 TMZ-treated cells (p < 0.05), and no TMZ effect was found in SF8628-treated cells. The study supports the suggestion that individual sensitivity to TMZ should be assessed when starting treatment.

The effect of valproic acid on SLC5A8 expression in gonad-intact and gonadectomized rat thymocytes.

BACKGROUND: Valproic acid (VPA) pharmacological mechanisms are related to the anti-inflammatory and anti-viral effects. VPA is a histone deacetylases inhibitor and serves a role in its immunomodulatory impacts. VPA has complex effects on immune cell's mitochondrial metabolism. The SLC5A8 transporter of short fatty acids has an active role in regulating mitochondrial metabolism. The study aimed to investigate whether SLC5A8 expresses the sex-related difference and how SLC5A8 expression depends on gonadal hormones, VPA treatment, and NKCC1 expression in rat thymocytes. METHODS: Control groups and VPA-treated gonad-intact and gonadectomized Wistar male and female rats were investigated (n = 6 in a group). The VPA 300 mg/kg/day in drinking water was given for 4 weeks. The SLC5A8 (Slc5a8 gene) and NKCC1 (Slc12a2 gene) RNA expressions were determined by the RT-PCR method. RESULTS: The higher Slc5a8 expression was found in the gonad-intact males than respective females (p = 0.004). VPA treatment decreased the Slc5a8 expression in gonad-intact and castrated males (p = 0.02 and p = 0.03, respectively), and increased in gonad-intact female rats compared to their control (p = 0.03). No significant difference in the Slc5a8 expression between the ovariectomized female control and VPA-treated females was determined (p > 0.05). VPA treatment alters the correlation between Slc5a8 and Slc12a2 gene expression in thymocytes of gonad-intact rats. CONCLUSION: VPA effect on the Slc5a8 expression in rat thymocytes is gender- and gonadal hormone-dependent.

Laryngeal carcinoma experimental model suggests the possibility of tumor seeding to gastrostomy site.

Some studies state that laryngeal squamous cell carcinoma (LSCC) is associated with possible direct tumor cell seeding to percutaneous endoscopic gastrostomy (PEG) site. However, there is a lack of experimental proof that LSCC tumor tissue can adhere and grow in distant sites. Therefore, we aimed to investigate the growth pattern of LSCC implants on chicken embryo chorioallantoic membrane (CAM) and evaluate possible associations between clinical course of the disease and behavior of experimentally implanted LSCC tumors. Our results show that implanted LSCC tissue survives on CAMs in 95% of cases while retaining essential morphologic characteristics and proliferative capacity of the original tumor. We identified the increased CAM vascularization, an infiltrative growth pattern of the implant and formation of distant isolated metastatic nodes on the CAMs. LSCC tumors with worse differentiation degree (G2 or G3) adhered to the experimental CAMs significantly better than G1. These results facilitate the understanding of tumor biology and allow hypothetisezing that dissemination and direct implantation of LSCC cells into the stomal wall during the pull PEG procedure might be possible.

The Effectiveness of Dichloroacetate on Human Glioblastoma Xenograft Growth Depends on Na+ and Mg2+ Cations.

The study's aim was to investigate the effectiveness of sodium dichloroacetate (NaDCA) or magnesium dichloroacetate (MgDCA) on adult U87 MG and pediatric PBT24 cell lines glioblastoma (GB) xenografts in a chicken chorioallantoic membrane (CAM) model. The study groups were: treated with 10 mM, 5 mM of NaDCA, and 5 mM, 2.5 mM of MgDCA, and controls. The U87 MG and PBT24 xenografts growth, frequency of tumor invasion into CAM, CAM thickening, and the number of blood vessels in CAM differed depending on the dichloroacetate salt treatment. NaDCA impact on U87 MG and PBT24 tumor on proliferating cell nunclear antigen (PCNA) and enhancer of zeste homolog 2 (EZH2) expression in the tumor was different, depending on the NaDCA dose. The 5 mM MgDCA impact was more potent and had similar effects on U87 MG and PBT24 tumors, and its impact was also reflected in changes in PCNA and EZH2 expression in tumor cells. The U87 MG and PBT24 tumor response variations to treatment with different NaDCA concentration on tumor growth or a contrast between NaDCA and MgDCA effectiveness may reflect some differences in U87 MG and PBT24 cell biology.

An Experimental Model of Human Recurrent Respiratory Papillomatosis: A Bridge to Clinical Insights.

OBJECTIVES/HYPOTHESIS: To investigate the growth pattern of recurrent respiratory papilloma (RRP) implants on chicken embryo chorioallantoic membranes (CAMs) and to evaluate possible associations between the clinical course of the disease and the behavior of experimentally implanted RRP tumors. STUDY DESIGN: Experimental study. METHODS: Fresh 172 RRP tissue samples from 12 patients were implanted onto chick embryo CAMs. Morphological and morphometric analysis of the experimental CAM and chorionic epithelium was performed. The microvascular network of the CAM with the RRP implant was investigated under the effect of fluoresceinated anionic dextran. The peculiarities of the clinical course of the disease were evaluated. RESULTS: The implanted RRP tissue samples survived on CAMs in 86% of cases, retaining their essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM and the chorionic epithelium, but none of the RRP implants crossed the basement membrane of the hosting CAM. A "crawling film" of acellular material with newly formed papilloma sprouts located on the outer chorionic epithelium of the CAM was detected. Direct association between a recurrence rate of RRP and the number of newly formed papilloma sprouts around the implanted tumor on CAMs was revealed. CONCLUSION: The chicken embryo CAM-based model is appropriate for investigations of RRP and facilitates the understanding of tumor biology and the clinical course of the disease, thus providing the basis for further research and acceleration of the identification and development of new specific therapeutic compounds that limit the spread and recurrence of RRP. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E914-E920, 2021.

The Anti-Tumorigenic Activity of Sema3C in the Chick Embryo Chorioallantoic Membrane Model.

Sema3C protein, a member of the class 3 family of secreted semaphorins, play an important role in tumor development by regulating cell proliferation, migration, invasion, and angiogenesis processes. Depending on the type and malignancy grade of the tumor, Sema3C function remains controversial. In this study, we constructed a stably overexpressing Sema3C glioblastoma cell line U87 MG and tested it on the chicken embryo chorioallantoic membrane (CAM) model with the aim to reveal Sema3C protein function on angiogenesis process in ovo. Our experiments showed that Sema3C not only affects angiogenesis of CAM by inhibiting neovascularization but also acts as an anti-tumorigenic molecule by hampering U87 MG cell invasion into mesenchyme. The effects of Sema3C on CAM were similar to the effects of anti-epileptic drug sodium valproate (NaVP). Both, anti-angiogenic and anti-tumorigenic activities of Sema3C were enhanced by the treatment of NaVP and, importantly, were not attributed to the cytotoxic effects. Our studies suggest that Sema3C could be a promising target for glioblastoma treatment.

The Importance of Gender-Related Anticancer Research on Mitochondrial Regulator Sodium Dichloroacetate in Preclinical Studies In Vivo.

Sodium dichloroacetate (DCA) is an investigational medicinal product which has a potential anticancer preparation as a metabolic regulator in cancer cells' mitochondria. Inhibition of pyruvate dehydrogenase kinases by DCA keeps the pyruvate dehydrogenase complex in the active form, resulting in decreased lactic acid in the tumor microenvironment. This literature review displays the preclinical research data on DCA's effects on the cell pyruvate dehydrogenase deficiency, pyruvate mitochondrial oxidative phosphorylation, reactive oxygen species generation, and the Na+-K+-2Cl- cotransporter expression regulation in relation to gender. It presents DCA pharmacokinetics and the hepatocarcinogenic effect, and the safety data covers the DCA monotherapy efficacy for various human cancer xenografts in vivo in male and female animals. Preclinical cancer researchers report the synergistic effects of DCA combined with different drugs on cancer by reversing resistance to chemotherapy and promoting cell apoptosis. Researchers note that female and male animals differ in the mechanisms of cancerogenesis but often ignore studying DCA's effects in relation to gender. Preclinical gender-related differences in DCA pharmacology, pharmacological mechanisms, and the elucidation of treatment efficacy in gonad hormone dependency could be relevant for individualized therapy approaches so that gender-related differences in treatment response and safety can be proposed.

Valproic Acid Inhibits NA-K-2CL Cotransporter RNA Expression in Male But Not in Female Rat Thymocytes.

OBJECTIVE: The NKCC1 is a recognized tumorigenesis marker as it is important for tumor cell proliferation, differentiation, apoptosis, and tumor progression. The study aim was to investigate the effect of sodium valproate (VPA) on thymus NKCC1 RNA expression. MATERIAL AND METHODS: Wistar rats, age 4 to 5 weeks, were investigated in the control and VPA-treated male and female gonad-intact and castrated groups. The treatment duration with VPA 300 mg/kg/d was 4 weeks. Rat thymus was weighted; its lobe was taken for the expression of NKCC1 RNA determined by the real-time polymerase chain reaction method. RESULTS: The RNA expression of the Slc12a2 gene was found to be significantly higher in the gonad-intact male control compared with the gonad-intact female control (P = .04). There was a gender-related VPA treatment effect on NKCC1 RNA expression in thymus: The Slc12a2 gene RNA expression level was found to be decreased in VPA-treated gonad-intact males (P = .015), and no significant VPA effects were found in the castrated males and in the gonad-intact and castrated females compared with the respective controls (P > .05). CONCLUSIONS: The study showed a gender-related difference in the NKCC1 RNA expression in rat thymus. The VPA decreases the NKCC1 expression in the thymus only in gonad-intact male rats. The NKCC1 RNA expression downregulation by VPA could be important for further VPA pharmacological studies in oncology.

Gender-Related Effect of Sodium Dichloroacetate on the Number of Hassall's Corpuscles and RNA NKCC1 Expression in Rat Thymus.

The aim was to investigate the effect of dichloroacetate (DCA) on thymus weight, Hassall's corpuscle number (HCs), and NKCC1 RNA expression in Wistar rats aged 4-5 weeks. They were investigated in the controls and DCA-treated gonad-intact and castrated males and females. The treatment lasted 4 weeks with DCA 200 mg/kg/day. At the end of the experiment, rat thymus was weighted, and its lobe was taken for the expression of NKCC1 RNA determined by the PCR method and of Hassall's corpuscles by immunohistochemistry. DCA caused a thymus weight decrease in DCA-treated gonad-intact rats of both genders as compared with their controls (p < 0.05), and no such impact was found in castrated DCA-treated males and females. DCA caused an increase of the HCs in gonad-intact males (p < 0.05), and no such increase in the DCA-treated gonad-intact females was found. There was gender-related difference in the HCs when comparing DCA-treated gonad-intact males and females: males showed significantly higher HCs (p < 0.05); no gender-related differences were found in the castrated DCA-treated groups. The Slc12a2 gene RNA expression level was found to be significantly decreased only in gonad-intact and in castrated DCA-treated males. The authors discuss the gender-related DCA effects on the thymus.

Sodium Dichloroacetate Pharmacological Effect as Related to Na-K-2Cl Cotransporter Inhibition in Rats.

The study objective was to investigate a possible sodium dichloroacetate (DCA) pharmacological mechanism causing an increase in diuresis in rats. The aim was to define characteristics of 24-hour urinary Na+, K+, Cl-, Ca2+, and Mg2+ excretion in Wistar male rats and to evaluate effect of a single-dose DCA and repeated DCA dosage on diuresis. Six control and 6 DCA-treated male rats aged 5 to weeks after a single DCA dose and repeated dosage were tested. The single DCA dose treatment caused a significantly higher 24-hour diuresis when compared to control (P < .05), and it was related to increased Cl-, Na+, and K+ urine excretion and a significant increase in Ca2+ and Mg2+ excretion (P < .05); after the repeated 4-week DCA dosage, the diuresis was not increased, but the excretion of the Na+, Cl-, Ca2+, and Mg2+ ions was significantly higher. Kidney immunohistochemistry has revealed that DCA continuous treatment results in an increase in the size of Henle loop thick ascending limb epithelial cells (P < .001). The study results show a significantly reduced RNA expression of Na-K-2Cl co-transporter (NKCC1) in thymus of 4-week DCA-treated rats (P < .03). The study data have indicated a possible mechanism of such pharmacological effect to be NKCC inhibition.

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.

Model of human recurrent respiratory papilloma on chicken embryo chorioallantoic membrane for tumor angiogenesis research.

We aimed to develop a chick embryo chorioallantoic membrane (CAM) model of recurrent respiratory papilloma (RPP) and to evaluate its morphological and morphometric characteristics, together with angiogenic features. Fresh RRP tissue samples obtained from 13 patients were implanted in 174 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up until 7 days after the implantation. Immunohistochemical analysis (34ßE12, Ki-67, MMP-9, PCNA, and Sambucus nigra staining) was performed to detect cytokeratins and endothelial cells and to evaluate proliferative capacity of the RRP before and after implantation on the CAM. The implanted RRP tissue samples survived on CAM in 73% of cases while retaining their essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (241-560%, p=0.001) and the chorionic epithelium (107-151%, p=0.001), while the number of blood vessels (37-85%, p=0.001) in the CAM increased. The results of the present study confirmed that chick embryo CAM is a relevant host for serving as a medium for RRP fresh tissue implantation. The CAM assay demonstrated the specific RRP tumor growth pattern after implantation and provided the first morphological and morphometric characterization of the RRP CAM model that opens new horizons in studying this disease.

The effect of dichloroacetate on male rat thymus and on thymocyte cell cycle.

The study aim was to investigate the effect of dichloroacetate (DCA) on thymus and the thymocyte cycle in rats. Wistar male gonad-intact and castrated rats (4-5 weeks) were investigated in the following groups: (1) control; (2) treated with DCA; and (3) treated with the DCA and sodium valproate (NaVP) combination. Rats were treated for 4 weeks with DCA 200 mg/kg/day alone and 300 mg/kg/day of NaVP plus 200 mg/kg/day of DCA (every second week, beginning with NaVP). After the experiment, the thymus was weighted, and the thymus lobe was taken for thymocyte flow cytometry. In gonad-intact rats, the thymus weight of the control was higher than in rats treated with DCA (P <0.001) or with the NaVP-DCA combination (P <0.04); a comparison of thymus weight between DCA- and NaVP-DCA-treated groups revealed a higher thymus weight loss in the DCA-treated group (P <0.03). Flow cytometry shows that DCA treatment increased the percentage of cells in the G2-M phase (P <0.03) and reduced in G1-G0 (P <0.02). The DCA treatment effect was determined only in gonad-intact but not in castrated rats. The authors discuss the possible DCA and NaVP interaction mechanisms.

Effect of Laryngeal Squamous Cell Carcinoma Tissue Implantation on the Chick Embryo Chorioallantoic Membrane: Morphometric Measurements and Vascularity.

BACKGROUND: The aim of this study was to develop chick embryo chorioallantoic membrane (CAM) model of laryngeal squamous cell carcinoma (LSCC) and to evaluate the morphological and morphometric characteristics and angiogenic features of it. METHODS: Fresh LSCC tissue samples obtained from 6 patients were implanted onto 15 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up to 4 days after the tumor implantation. Immunohistochemical analysis (34ßE12, CD31, and Ki67 staining) was performed to detect cytokeratins and tumor endothelial cells and to evaluate the proliferative capacity of the tumor before and after implantation on the CAM. RESULTS: The implanted LSCC tissue samples survived on the CAM in all the experiments and retained the essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (103-417%, p = 0.0001) and the chorionic epithelium (70-140%, p = 0.0001) and increase in number of blood vessels (75-148%, p = 0.0001) in the CAM. CONCLUSIONS: This study clarifies that chick embryo CAM is a relevant assay for implanting LSCC tissue and provides the first morphological and morphometric characterization of the LSCC CAM model that opens new perspectives to study this disease.