Potential role of P2X7R in esophageal squamous cell carcinoma proliferation.

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5'-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.

Induced pluripotent stem cells from patients with focal cortical dysplasia and refractory epilepsy.

Focal cortical dysplasia (FCD) is caused by numerous alterations, which can be divided into abnormalities of the cortical architecture and cytological variations; however, the exact etiology of FCD remains unknown. The generation of induced pluripotent stem cells (iPSCs) from the cells of patients with neurological diseases, and their subsequent tissue­specific differentiation, serves as an invaluable source for testing and studying the initial development and subsequent progression of diseases associated with the central nervous system. A total of 2 patients demonstrating seizures refractory to drug treatment, characterized as FCD Type IIb, were enrolled in the present study. Fibroblasts were isolated from residual skin fragments obtained from surgical treatment and from brain samples obtained during surgical resection. iPSCs were generated following exposure of fibroblasts to viral vectors containing POU class 5 homeobox 1 (OCT4), sex determining region Y­box 2 (SOX2), Kruppel­like factor 4 and c­MYC genes, and were characterized by immunohistochemical staining for the pluripotent markers homeobox protein NANOG, SOX2, OCT4, TRA1­60 and TRA1­81. The brain samples were tested with antibodies against protein kinase B (AKT), phosphorylated­AKT, mechanistic target of rapamycin (mTOR) and phosphorylated­mTOR. Analysis of the AKT/mTOR pathway revealed a statistically significant difference between the cerebral tissues of the two patients, which were of different ages (45 and 12 years old). Clones with the morphological features of embryonic cells were detected on the 13th day and were characterized following three subcultures. The positive staining characteristics of the embryonic cells confirmed the successful generation of iPSCs derived from the patients' fibroblasts. Therefore, the present study presents a method to obtain a useful cellular source that may help to understand embryonic brain development associated with FCD.

Laboratory methods and biomarkers in the evaluation of bisphosphonate effects on body tissues: a literature review.

Bisphosphonates are extensively used to treat bone metabolism disorders; meanwhile, these drugs have important side effects such as jaw osteonecrosis and femoral fracture. Therefore, studies have been conducted aimed at better understanding their mechanism of action and determining a course of action to avoid these side effects. We present here a literature review focusing on the laboratory methods available for investigating bisphosphonate effects on body tissues. There are many different methods available for this purpose, but the appropriate application of these methods and knowledge of their limitations are crucial for understanding the effects of bisphosphonates.

Inhibition of phosphatidylinositol-3 kinase γ reduces pruriceptive, inflammatory, and nociceptive responses induced by trypsin in mice.

This study investigated the effects of pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K)γ in the pruriceptive, inflammatory, and nociceptive responses induced by trypsin in mice. The animals were orally treated with the selective PI3Kγ inhibitor AS605240 (0.3-30 mg/kg) 30 minutes beforehand. In separate groups, AS605240 was given by intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes. The control groups received saline at the same schedules. The effects of PI3K blocking were assessed in different experimental assays. The oral treatment with AS605240 produced a marked reduction of scratching behavior elicited by trypsin. Moreover, AS605240 (1mg/kg) was able to produce a partial but significant inhibition of the scratching bouts elicited by CP 48/80. Interestingly, the i.c.v. and i.t. injection of AS605240 also reduced trypsin-induced itching. The oral administration of AS605240 was found effective in producing a significant and dose-dependent reduction of trypsin-induced paw edema and tumor necrosis factor α production, as well as the neutrophil recruitment, according to myeloperoxidase activity assessment. Likewise, oral AS605240 (1mg/kg) promoted a significant reduction of spontaneous nociception induced by trypsin in the mouse paw. In contrast, the oral administration of AS605240 did not significantly modify capsaicin-evoked nociception, although this inhibitor was effective when dosed by i.c.v. route. Noteworthy, AS605240 (1mg/kg) was able to prevent c-Fos and phospho-Akt immunopositivity at the spinal cord of trypsin-injected mice, either into the back of the neck or the paws. To conclude, PI3Kγ inhibition might well represent a valuable alternative for treating inflammatory and painful conditions, as well as pruritus.

Immunohistochemical expression of DNA methyltransferases 1, 3a and 3b in oral leukoplakias and squamous cell carcinomas.

UNLABELLED: Over-expression of DNA methyltransferases DNMT1, DNMT3a and DNMT3b has been reported in various cancers and precancerous lesions. OBJECTIVE: To investigate DNMT1, DNMT3a and DNMT3b enzymes in oral squamous cell carcinoma (SCC) and leukoplakia, and their relationship with histopathologic/clinical parameters. STUDY DESIGN: Immunohistochemistry was carried out to evaluate the three DNMTs in 60 samples of oral SCC and 37 samples of oral leukoplakia. RESULTS: DNMT3a immunoreactivity in the three groups of oral SCC (39.8%) was significantly higher than in control (22.6%) (ANOVA, Student-Newman-Keuls test, P<0.05), but not when compared to oral leukoplakia groups (28.2%). For DNMT1 and DNMT3b, there were no statistically significant differences between oral SCC groups (65% and 74.7%), oral leukoplakia groups (68.3% and 70.9%) and control (65.4% and 76.5%). There was a significantly higher mean percentage of DNMT1 immunoreactivity in non-smokers (ANOVA, P=0.048), and a higher DNMT3a immunoreactivity in alcohol users (ANOVA, P=0.01). CONCLUSIONS: Higher DNMT3a immunopositivity may be associated with oral SCC and alcohol use, whilst lower levels of DNMT1 may be related with smoking habit. However, there was a significantly higher mean percentage of DNMT1 immunoreactivity in non-smokers (ANOVA, P=0.048), and a higher DNMT3a immunoreactivity in alcohol users (ANOVA, P=0.010).

Effects of the compounds MV8608 and MV8612 obtained from Mandevilla velutina in the model of hemorrhagic cystitis induced by cyclophosphamide in rats.

Hemorrhagic cystitis (HC) is a common side effect observed in patients under chemotherapy with cyclophosphamide (CYP). The urotoxic side effects of CYP are attributed to the metabolic compound acrolein, and can be partially prevented by the uroprotector agent 2-mercaptoethene sulfate (Mesna). The present study analyzed the anti-inflammatory and the antinociceptive effects of compounds MV8608 and MV8612 obtained from Mandevilla velutina in the rat model of CYP-induced HC. Male Wistar rats were used (six to eight per group, 220-250 g). HC was induced by a single administration of CYP (100 mg/kg, ip). Three behavioral parameters--breathing rate, closing of the eyes, and specific posture--were used as nociception indexes, and scored at different time intervals (15-180 min) after cystitis induction. As inflammatory parameters, hemorrhage presence, edema formation, and bladder weight were determined at 24 h after CYP administration. The neutrophil migration was assessed by means of myeloperoxidase (MPO activity), 4 h after cystitis induction. As expected, Mesna treatment was able to reduce in a significant manner all the inflammatory and the nociceptive parameters induced by CYP. Of note, the administration of MV8608 significantly inhibited the hemorrhage formation and the neutrophil recruitment, while the MV8612 treatment markedly reduced the bladder weight, without interfering with neutrophil influx. Interestingly, the treatment with either MV8608 or MV8612 markedly reduced the nociceptive responses. The present results clearly indicate that MV8608 and MV8612 might represent important alternatives to prevent side effects, especially the nociception, following chemotherapy with CYP.