Clinical and biochemical assessment of the effect of glutamine in management of radiation induced oral mucositis in patients with head and neck cancer: Randomized controlled clinical trial.

BACKGROUND: This study aimed to evaluate the effect of oral glutamine suspension on salivary levels of transforming growth factor beta 1 (TGF-ß1), a cytokine involved in inflammation and Tumor progression, and the severity of radiation-induced oral mucositis (RIOM) in head and neck cancer patients. This is the first study to investigate the impact of glutamine on TGF-ß1 levels in head and neck cancer patients with radiation induced oral mucositis (RIOM). METHODS: In this randomized controlled clinical trial, 50 HNC patients were enrolled and received either glutamine oral suspension or maltodextrin as a placebo from the baseline of RIOM to the end of radiotherapy. Salivary TGF-ß1 levels were measured at baseline and after treatment. Also, RIOM was assessed using the World Health Organization (WHO) Oral Toxicity Scale, the Oral Mucositis Assessment Scale (OMAS), the Pain Visual Analog Scale (Pain-VAS), the incidence of opioid use, and body mass index (BMI). RESULTS: Glutamine significantly reduced salivary TGF-ß1 levels and improved RIOM symptoms, such as pain, opioid use, and weight loss. The reduction of TGF-ß1 levels was associated with the improvement of RIOM severity. CONCLUSION: Glutamine may modulate the inflammatory response and enhance wound healing in RIOM by decreasing salivary TGF-ß1 levels. These findings support the use of glutamine as a potential intervention for RIOM and nutritional support for improving radiation sensitivity. TRIAL REGISTRATION: This study was registered on clinicalTrials.gov with identifier no. NCT05856188.

Papel de TGFß-1 na regulação da expressão de MMPs seus inibidores (TIMPs e Reck) em modelo de carcinoma mamário humano: análise funcional de RECK e sua correlação com dados clínico-patológicos / Role of TGFß-1 as a common regulator of MMPs and their inhibitors (TIMPs e RECK) in human breast cancer cell model: functional analysis of RECK and its correlation with clinical-pathological

A causa de morte da maioria das pacientes com câncer de mama se deve à doença metastática desenvolvida a partir do tumor primário. A degradação dos componentes da matriz extracelular (MEC), um dos principais eventos do processo metastático, é regulada pelo balanço entre as atividades das metaloproteinases de matriz (MMPs) e dos seus inibidores, tanto os inibidores teciduais (TIMPs) como o inibidor associado à membrana (RECK). Contudo, ainda existe pouca informação sobre os mecanismos moleculares responsáveis pela manutenção deste balanço. No presente trabalho, foi investigado o envolvimento de TGF-ß1 (Transforming Growth Factor-ß1), uma citocina multifuncional é capaz tanto de inibir o crescimento celular, quanto de promover invasão e metástase, dependendo do estadiamento e do tipo de tumor, na regulação da expressão de MMPs, TIMPs e RECK, em modelo de câncer de mama. Primeiramente, examinou-se os níveis de expressão de mRNA das isoformas e receptores de TGF-ß, em um painel de cinco linhagens de carcinoma mamário humano, com diferentes potenciais invasivos e metastáticos, por qRT-PCR. Os resultados obtidos demonstraram uma correlação positiva entre a expressão dessas moléculas, e a progressão do caráter invasivo e metastático celular. Em seguida, a linhagem altamente invasiva, MDA-MB-231, foi tratada com diferentes concentrações de TGF-ß1 recombinante. Esta citocina foi capaz de modular a expressão gênica de MMPs (MMP-2 e MMP-9) e de seus inibidores (TIMP- 2 e RECK). Tanto ERK½, quanto p38MAPK mostraram-se envolvidas neste mecanismo. Foi demonstrado que a inibição da atividade de ERK½ alterou a expressão das proteínas MMP-9, TIMP-2 e RECK, enquanto o bloqueio de p38 MAPK afetou os níveis protéicos de MMP-2 e TIMP-2. O aumento do potencial migratório e invasivo da linhagem MDA-MB-231, induzido por TGF-ß1, mostrou-se também dependente da atividade de MMPs, ERK½ e p38MAPK. Dada a ausência de informações sobre o papel de RECK em modelo mamário, a função deste inibidor de MMPs também foi investigada. Primeiramente, analisou-se a expressão de RECK ao longo do desenvolvimento da mama e, posteriormente, em 1040 amostras tumorais de mama humana, através da metodologia de Tissue Microarray, tendo sido possível demonstrar que a alta expressão de RECK associa-se a menor tempo de sobrevida global e livre de doença em 10 anos. Os resultados obtidos indicaram que a expressão da proteína RECK, em oposição ao verificado em outros tipos de tumores, está relacionada ao fenótipo mais agressivo de tumores de mama. Entretanto, a análise funcional de RECK, realizada por meio da utilização de vetores shRNA específicos para a inibição desta proteína, demonstrou que RECK também atua como um inibidor de invasão celular e da expressão de MMP-9, na linhagem MDA-MB-231. Em conjunto, os resultados obtidos neste trabalho contribuíram para a elucidação dos mecanismos moleculares de regulação de RECK, por clássicas moléculas associadas ao processo de tumorigênese (TGF-ß1 e MAPKs), bem como para o esclarecimento de suas funções em modelo mamário, sugerindo-o como mais um promissor candidato a marcador prognóstico e alvo molecular para a terapia do câncer de mama

The metastatic disease is the main mortality cause of breast cancer patients. The metastatic process involves a complex cascade of events, including the organized breakdown of the extracellular matrix (ECM) compounds. The degradation of ECM is tightly regulated by the balance between the activities of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors (TIMPs) and the membrane-associated inhibitor (RECK). Among the several molecules released and activated by ECM remodeling, TGF-ß1 (Transforming Growth Factor-ß1) is a multifunctional cytokine able to regulate both cell growth inhibition and invasion and metastasis promotion, depending on the tumor stage and type. Since the molecular mechanisms involved in the ECM remodeling control are still not completed understood, in this study, we investigated the involvement of TGF-ß1 in regulating of MMPs, TIMPs and RECK expression, in the breast cancer model. By qRT-PCR, we first examined the gene expression levels of TGF-ß isoforms and receptors, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. Our results suggest a positive correlation between the mRNA expression of these molecules and the breast cancer progression. Moreover, the highly invasive breast cancer cell line MDA-MB-231 was treated with different concentrations of recombinant TGF-ß1. We described that this cytokine was able to modulate the gene expression of MMPs (MMP-2 and MMP-9) and MMPs inhibitors (TIMP-2 and RECK) at both the mRNA and protein levels, with ERK½ and p38 MAPK being involved in this molecular mechanism. However, while ERK½ activity inhibition altered MMP-9, TIMP-2 and RECK expression, the p38 MAPK blockage affected the protein levels of MMP-2 and TIMP-2. Finally, we reposted that the TGF-ß1-enhanced migration and invasion capacities of MDA-MB- 231 cells were blocked by MMPs, ERK½ and p38 MAPK inhibitors. Analysis of the RECK function in the breast model was also an objective of this study. We analyzed RECK expression during mammary gland development. We evaluated the RECK protein profile in 1040 breast tumor tissue samples using Tissue Microarray assays. We demonstrated that high expression levels of RECK were associated with shorter overall and disease-free survival in 10 years. Moreover, we verified that RECK is a biomarker of poor prognosis mainly for patients diagnosed with less aggressive breast tumor. Therefore, in contrast to other tumor types, our results indicate that high protein expression levels of RECK are related to a more aggressive phenotype. In fact, the RECK functional analysis, performed by using of shRNA vectors, showed that RECK function remains as an inhibitor of cellular invasion and MMP-9 expression, in MDA-MB-231 cells. Taken together, our results contribute to better understanding of the molecular mechanisms associated to RECK regulation by TGF-ß1 and MAPK as well as to clarify its role in breast model. Thus, we suggests RECK as a new and promising prognostic marker and molecular target candidate for breast cancer therapy

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

Quantitative assessment of the kinetics of growth factors release from platelet gel.

BACKGROUND: The time course of the release of growth factors from platelet (PLT) gels has not been thoroughly studied and should be elucidated for a better standardization of the clinical use of these products. STUDY DESIGN AND METHODS: Release of PLT-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-1 (IGF-1) was determined 5, 60, 120, and 300 minutes after PLT gel formation. Control experiments where PLT gel was removed and, afterward, exogenous thrombin was added, were also performed. Protein profiles of the PLT concentrates and of the releasates were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Mean PDGF-AB, TGF-beta1, EGF, and VEGF concentration increased to 76, 114, 3.7, and 0.8 ng per mL, respectively, in the presence of the PLT gel, but remained at approximately 28, 30, 0.28, and 0.34 ng per mL, respectively, when the PLT gel was removed after formation. IGF-1 content remained unchanged (approx. 80 ng/mL). SDS-PAGE analysis showed that several PLT proteins disappear during PLT gel formation and that the protein patterns of the releasates were undistinguishable at the different time points. CONCLUSION: There is a gradual and fast release of PDGF-AB, TGF-beta1, EGF, and VEGF from PLT gel for at least 60 to 300 minutes after gel formation, whereas the IGF releasate concentration remains unchanged. This study may provide useful information to improve clinical applications of PLT gels and to design improved blood-derived biomaterials with controlled release of growth factors.

Quantitative assessment of growth factors in reaming aspirate, iliac crest, and platelet preparation.

Large bony defects and non-unions are still a complication in trauma and orthopedic surgery. Treatment strategies include the use of autogenous materials (iliac crest), allogenic bone, bone substitutes, and currently stimulation with growth factors such as BMP-2, BMP-7 or the growth factors containing platelet-rich plasma (PRP). Another source of bone graft material might be the cuttings produced during intramedullary reaming. The aim of this study was to compare the quantity of various growth factors found within iliac crest, bony reaming debris, reaming irrigation fluid, and platelet-rich plasma. Iliac crest and reaming debris and irrigation samples were harvested during surgery. PRP was prepared from blood. The growth factors in the bony materials (iliac crest or reaming debris) and of the liquid materials (platelet-poor plasma (PPP), platelet-rich plasma (PRP) or reaming irrigation) were compared. Elevated levels of FGFa, PDGF, IGF-I, TGF-beta1 and BMP-2 were measured in the reaming debris as compared to iliac crest curettings. However, VEGF and FGFb were significantly lower in the reaming debris than from iliac crest samples. In comparing PRP and PPP all detectable growth factors, except IGF-I, were enhanced in the platelet-rich plasma. In the reaming irrigation FGFa (no measurable value in the PRP) and FGFb were higher, but VEGF, PDGF, IGF-I, TGF-beta1 and BMP-2 were lower compared to PRP. BMP-4 was not measurable in any sample. The bony reaming debris is a rich source of growth factors with a content comparable to that from iliac crest. The irrigation fluid from the reaming also contains growth factors.