Enxerto de látex natural na cicatrização de esclerectomias lamelar e penetrante em coelhos

The objective of study was to investigate the effects of natural latex with 0.1% of polylysine on lamellar and penetrating scleroctomies in rabbits. Two groups of twelve rabbits each (lamellar GI and penetrating GII) were studied. Scleral square incisions near the limbus were performed on the left eye of each animal. The latex biomembrane was fixed to the recipient sclera and it covered with a conjunctival flap. The clinical evaluations were followed for 60 days. Aplannation tonometry, binocular indirect ophthalmoscopy and slit-lamp biomicroscopy were performed during evaluation. Bright field microscopy and polarization microscopy were employed. Blepharospasm, graft infection, mucoid ocular discharge and chemosis were not observed in either treatment group. The conjunctival hyperemia varied from moderate to hardly noticeable. The postoperative IOP was not statiscally significant, comparing to the preoperative IOP, for GI and GII. The histopathology by polarization microscopy showed that the neoformed tissue was primarily dependent on adjacent vascularized tissues and was constituted by collagen type III. Both groups presented optimum graft adhesion to the receiving sclera. The natural latex biomembrane with 0.1% polylysine constitutes a new alternative for scleral reconstruction. Furthermore, this is a durable material, easy to obtain and manipulate.Este trabalho teve como objetivo investigar os efeitos do látex natural com polilisina a 0,1% na cicatrização de esclerectomias lamelar e penetrante em coelhos. Foram estudados dois grupos de 12 coelhos (GI - lamelar e GII - penetrante). As esclerectomias foram realizadas no olho esquerdo de cada animal. A biomembrana de látex foi fixada à esclera receptora e foi recoberta com conjuntiva bulbar. As avaliações clínicas foram realizadas durante 60 dias. Para tal, empregaram-se a tonometria de aplanação, a oftalmoscopia indireta binocular e a biomicroscopia em lâmpada de fenda. Realizou-se análise histopatológica das escleras em microscopia de luz e sob luz polarizada. Não houve blefarospasmo, secreção ocular mucóide, quemose ou sinais de infecção do enxerto em ambos os grupos. A hiperemia conjuntival variou de moderada a tênue. Não houve diferença estatisticamente significativa entre a pressão intraocular do período pós-operatório e do período pré-operatório, para o GI e o GII. A histopatologia por microscopia de polarização demonstrou que o tecido neoformado foi oriundo de tecidos vascularizados adjacentes e constituídos por colágeno do tipo III. Os dois grupos apresentaram ótima adesão do enxerto de látex à esclera receptora. A biomembrana de látex natural com polilisina a 0,1% representa uma alternativa para a reconstrução escleral, além de apresentar fácil obtenção, manuseio e durabilidade

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.FEDER through POFC – COMPETE and by Fundação para a Ciência e Tecnologia through projects PEst-OE/BIA/UI4050/2014 and FCT ANR/BEX-BCM/0175/201

Predictions for the future of kallikrein-related peptidases in molecular diagnostics

Kallikrein-related peptidases (KLKs) form a cancer-related ensemble of serine proteases. This multigene family hosts the most widely used cancer biomarker that is PSA-KLK3, with millions of tests performed annually worldwide. The present report provides an overview of the biomarker potential of the extended KLK family (KLK1-KLK15) in various disease settings and envisages approaches that could lead to additional KLK-driven applications in future molecular diagnostics. Particular focus is given on the inclusion of KLKs into multifaceted cancer biomarker panels that provide enhanced diagnostic, prognostic and/or predictive accuracy in several human malignancies. Such panels have been described so far for prostate, ovarian, lung and colorectal cancers. The role of KLKs as biomarkers in non-malignant disease settings, such as Alzheimer’s disease and multiple sclerosis, is also commented upon. Predictions are given on the challenges and future directions regarding clinically oriented KLK research

Overexpression of matrix-metalloproteinase-9 in human breast cancer: a potential favourable indicator in node-negativeatients

Matrix metalloprotease-9 (MMP-9; 92 kDa type IV collaganase, gelatinase B) is regarded as, important for degradation of the basement membrane and extracellular matrix during cancer invasion and other tissue-remodelling events. In this study we evaluate the prognostic value of MMP-9, by immunoperoxidase staining in a series of 210 breast cancer tissues. The results were quantitated using the HSCORE system, which consider both staining intensity and the percentage of cells stained at given intensities. MMP-9 status was compared with the concentration of cytosolic Cathepsin-D and with other established prognostic factors, in terms of disease free survival and overall survival. The median follow-up period was 62 months. MMP-9 staining was observed primarily in cancer cells, and to a lesser degree in surrounding stromal cells. MMP-9 expression was not detected in normal breast tissue. Levels of MMP-9 expression below the cut-off point were more frequently observed in larger (P = 0.014), invasive ductal histologic (P = 0.037), progesterone receptor (PR)-negative and PR-strong positive tumours (P< 0.001), as well as samples belonging to patients with stage III-IV disease (P = 0.009) and age 45–55 years (P = 0.011). In univariate analysis, node-negative breast cancer patients with tumors positive for MMP-9 had a considerable reduction in risk for relapse (RR = 0.45;P = 0.039) or death (RR = 0.32;P = 0.009). Multivariate analysis indicated that MMP-9 status was an independent favourable predictor of OS (RR = 0.47;P = 0.034) in node-negative but not in node-positive patients. Our results suggest that MMP-9 may be an independent favourable prognostic factor in node-negative breast cancer patients. The overexpression of MMP-9 in breast cancer may be also used as a marker to subdivide node negative breast cancer patients in order to determine the optimal treatment modality. © 2001 Cancer Research Campaign http://www.bjcancer.co

Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients

Background: Dysregulated expression of Kallikrein-related peptidase 6 (KLK6) is a common feature for many human malignancies and numerous studies evaluated KLK6 as a promising biomarker for early diagnosis or unfavorable prognosis. However, the expression of KLK6 in carcinomas derived from mucosal epithelia, including head and neck squamous cell carcinoma (HNSCC), and its mode of action has not been addressed so far. Methods: Stable clones of human mucosal tumor cell lines were generated with shRNA-mediated silencing or ectopic overexpression to characterize the impact of KLK6 on tumor relevant processes in vitro. Tissue microarrays with primary HNSCC samples from a retrospective patient cohort (n = 162) were stained by immunohistochemistry and the correlation between KLK6 staining and survival was addressed by univariate Kaplan-Meier and multivariate Cox proportional hazard model analysis. Results: KLK6 expression was detected in head and neck tumor cell lines (FaDu, Cal27 and SCC25), but not in HeLa cervix carcinoma cells. Silencing in FaDu cells and ectopic expression in HeLa cells unraveled an inhibitory function of KLK6 on tumor cell proliferation and mobility. FaDu clones with silenced KLK6 expression displayed molecular features resembling epithelial-to-mesenchymal transition, nuclear β-catenin accumulation and higher resistance against irradiation. Low KLK6 protein expression in primary tumors from oropharyngeal and laryngeal SCC patients was significantly correlated with poor progression-free (p = 0.001) and overall survival (p < 0.0005), and served as an independent risk factor for unfavorable clinical outcome. Conclusions: In summary, detection of low KLK6 expression in primary tumors represents a promising tool to stratify HNSCC patients with high risk for treatment failure. These patients might benefit from restoration of KLK6 expression or pharmacological targeting of signaling pathways implicated in EMT

Effect of antineoplastic agents on the expression of human telomerase reverse transcriptase b plus transcript in MCF-7 cells

Abstract Objectives: To evaluate the effect of antineoplastic agents on the expression of human telomerase reverse transcriptase (hTERT) splice variants in MCF-7 cells. Design and methods: We have developed a luminometric hybridization assay for hTERT beta plus transcript. MCF-7 cells were isolated before and after treatment with antineoplastic agents. A combination of nested RT-PCR and the developed luminometric hybridization assay was used for the specific detection of hTERT beta plus transcript in treated and untreated MCF-7 cells. Amplification of all hTERT splicing variants by nested PCR in the same samples was also performed. Results: MCF-7 cells treated with taxol and etoposide were found positive for all hTERT splicing variants, while the expression of hTERT beta plus transcript did not differ significantly before and after exposure. MCF-7 cells treated with doxorubicin and 5-fluorouracil did not express any of hTERT splicing variants. In the presence of cisplatin, three splicing variants of hTERT were detected. Conclusions: The developed hybridization assay is highly sensitive and specific for the detection of hTERT beta plus transcript in clinical samples

Use of Acrylic Resin to Fill the Orbital Cavity after Exenteration in a Dog with Conjunctival Hemangiosarcoma

Background: The hemangiossarcoma (HSA) is a malignant tumor originated from the alterations of vascular endothelial cells. As it has an aggressive behavior, it is indicated, as initial treatment, wide surgical excision, such as the exenteration, which results in the surgical removal of the ocular bulb and adjacent tissues. The referred technique has as a result a concave orbit and aesthetically unacceptable. Therefore, various materials, used as orbital implants, have been studied and used in several species. Thus, it was aimed to report the use of the polymethylmethacrylate (PMMA) to fill the orbital cavity after exenteration in a dog with conjunctival HSA.Case: A 10-year-old male Pitbull dog was assisted, with a clinical history of growth of a reddish tissue in the left eye, causing constant hemorrhage, with an evolution of two months. At ophthalmic examination of the left eye, in the temporal bulbar conjunctive was found a reddish neoformation, with an irregular surface, measuring approximately 4x2x2 cm. The biopsy and aspiration cytology of the neoformation, revealed cells that inferred that they were those of conjunctival HSA. The hemogram revealed normocytic anemia; the biochemical profile was with the standards of normality and no metastasis were found in the ultrasonography and X-ray. The treatment of choice was the exenteration. Initially, the palpebral borders were approximated using continuous simple suture wit monofilament nylon thread. An incision was made in the skin, along the orbital rim and then was performed a rhombus dissection of the conjunctive and all the extraocular muscles. Next, the eye globe, together with the neoplasia, soft tissues of the orbital cavity and third eyelid were removed. The PMMA was obtained from a mixture of the powder (polymer) and of the liquid (monomer) in the ratio of 1:1 in a sterile recipient, in order to obtain a liquid suspension. Still in its paste-like form, the PMMA was molded in the orbit itself without going beyond its limits, filling in. At the moment of the polymerization of the PMMA, the site was irrigated with cold sterile saline solution for the reduction of the temperature caused by the thermal reaction. For the closing of the conjunctive, a continuous simple suture was performed, using 910 4-0 polyglactin thread and, in the suture of the skin, simple interrupted stiches were used with 3-0 monofilament nylon thread. After the surgery, tramadol hydrochloride was administered at a dose of 2 mg/kg/SC and meloxicam at a dose of 0.1 mg/kg/SC.  In the post-operative phase, the animal was treated with ampicillin suspension at a dose of 20 mg/kg/TID/PO during 10 days; metronidazole at a dose of 15 mg/kg /BID/PO during 10 days and the application of 0.5% chloramphenicol ocular ointment, 10.000UI of retinyl acetate and 2.5% amino acids on the stiches, SID, until their removal. After nine months, the owner reported a satisfactory result in relation to the aesthetic aspect, denying the presence of any ocular alterations or discomfort of the animal.Discussion: The use of the PMMA implant proved to be an excellent alternative to fill the anophthalmic cavity after exenteration in a dog with conjunctival HSA, seen as it provided, most importantly, the maintenance of the orbital volume, granting an adequate aesthetic aspect, already established among some authors, without the presence of secretion, inflammation and local infection.  It is worth emphasizing that, despite the patient in question having been treat with a broad-spectrum antibiotic after the surgery, in other reports, the absence of local infection was associated to the high temperature that the PMMA reaches during the exothermic reaction in the polymerization stage. Furthermore, the referred implant is cost-effective, offers easy molding to the orbit and absence of extrusion. In light of this, it is therefore suggested that the referred implant is a pertinent alternative for use in similar cases

UCP2 and PRMT1 are key prognostic markers for lung carcinoma patients

Cancer cells have developed unique strategies to meet their high energy demand. Therefore, they have established a setting of Ca2+-triggered high mitochondrial activity. But mitochondrial Ca2+ uptake has to be strictly controlled to avoid mitochondrial Ca2+ overload that would cause apoptotic cell death. Methylation by protein arginine methyl transferase 1 (PRMT1) desensitizes the mitochondrial Ca2+ uptake machinery and reduces mitochondrial Ca2+ accumulation in cancer cells. In case of PRMT1- driven methylation, proper mitochondrial Ca2+ uptake is reestablished by increased activity of uncoupling protein 2 (UCP2), pointing to an importance of these proteins for cancer cell survival and activity. Accordingly, in this study we investigated the impact of UCP2 and PRMT1 on the fate of human lung cancer cells (A549, Calu-3 and H1299) as well as on patients suffering from lung carcinoma. We show that combined overexpression of UCP2 and PRMT1 significantly enhances viability, proliferation as well as mitochondrial respiration. In line with these findings, the overall survival probability of lung carcinoma patients with high mRNA expression levels of UCP2 and PRMT1 is strongly reduced. Furthermore, analysis via The Cancer Genome Atlas (TCGA) reveals upregulation of both proteins, UCP2 and PRMT1, as common feature of various cancer types. These findings suggest that proper mitochondrial Ca2+ uptake is essential for devastating tumor growth, and highlight the importance of a tightly controlled mitochondrial Ca2+ uptake to ensure proper ATP biosynthesis while avoiding dangerous mitochondrial Ca2+ overload. By that, the study unveils proteins of the mitochondrial Ca2+ uptake as potential targets for cancer treatment. Copyright: Sokolowski et al