Enhancing scaffold-free spheroid models: 3D cell bioprinting method for metastatic HSC3-Oral squamous carcinoma cell line.

3D in vitro systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of in vivo-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).

GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment.

BACKGROUND AIMS: Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. METHODS: hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. RESULTS: hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. CONCLUSIONS: hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

Arsenic Trioxide Triggers Apoptosis of Metastatic Oral Squamous Cells Carcinoma with Concomitant Downregulation of GLI1 in Hedgehog Signaling.

Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and the inhibition of the Hedgehog (HH) pathway in OSCC. Initially, ATO cytotoxicity was assessed in a panel of cell lines. Cell viability, cell cycle, death patterns, and cell morphology were analyzed, as well as the effect of ATO on the expression of HH pathway components. After the cytotoxic assay, HSC3 cells were chosen for all in vitro assays. ATO increased apoptotic cell death and nuclear fragmentation in the sub-G1 cell cycle phase and promoted changes in cell morphology. In addition, the reduced expression of GLI1 indicated that ATO inhibits HH activity. The present study provides evidence of ATO as an effective cytotoxic drug for oral cancer treatment in vitro.

Inhibition of CAL27 Oral Squamous Carcinoma Cell by Targeting Hedgehog Pathway With Vismodegib or Itraconazole.

Oral Squamous Cell Carcinoma (OSCC) presents an important challenge for the health systems worldwide. Thus, unraveling the biological mechanisms involved in OSCC pathogenesis is essential to the discovery of new drugs with anticancer potential. The Hedgehog (HH) pathway has shown promising results as a therapeutic target both in vitro and in vivo. This study aimed to investigate the effects of vismodegib and itraconazole on the expression of Hedgehog (HH) genes (PTCH1, SMO, and GLI1), cell cycle and cell death in OSCC cells. Alamar Blue assay was used to assess the cytotoxicity of vismodegib and itraconazole in a panel of oral cancer cell lines, including CAL27. The expression of HH signaling components after treatment with vismodegib and itraconazole, at concentrations of 25 or 50 µg/ml was evaluated by qPCR. Cell cycle and apoptosis were evaluated by flow cytometry after 72 h treatment with 50 µg/ml of vismodegib or itraconazole. HH signaling was activated in OSCC cell lines CAL27, SCC4, SCC9, and HSC3. Vismodegib and itraconazole significantly reduced CAL27 cell viability after 48 h of treatment. Gene expression of PTCH1, SMO, and GLI1 decreased in response to 24 h of treatment with vismodegib or itraconazole. Furthermore, CAL27 cells exhibited alterations in morphology, cell size, and cellular granularity. An increase in the DNA fragmentation was observed after treatment and both inhibitors induced apoptosis after 72 h. In conclusion, SMO inhibitors vismodegib and itraconazole demonstrably reduced the expression of HH genes in CAL27 OSCC cell line. In addition, treatment with vismodegib and itraconazole reduced cellular viability and altered the morphology of CAL27 cells, and also induced apoptosis.

ß-Lapachone and its iodine derivatives cause cell cycle arrest at G2/M phase and reactive oxygen species-mediated apoptosis in human oral squamous cell carcinoma cells.

ß-Lapachone is a natural naphthoquinone originally obtained from the bark of the purple Ipe (Tabebuia avellanedae Lor, Bignoniaceae) and its therapeutic potential in human cancer cells has been evaluated in several studies. In this study, we examined the effects of ß-lapachone and its 3-iodine derivatives (3-I-α-lapachone and 3-I-ß-lapachone) on cell proliferation, cell death, and cancer-related gene expression in human oral squamous cell carcinoma cells. ß-Lapachone and its 3-iodine derivatives showed potent cytotoxicity against different types of human cancer cell lines. Indeed, treatment with these compounds induced cell cycle arrest at G2/M phase, followed by internucleosomal DNA fragmentation, and caused significant increases in phosphatidylserine externalization, caspase-8 and -9 activation, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, and apoptotic cell death morphology. The apoptosis induced by the compounds was prevented by pretreatment with a pan-caspase inhibitor (Z-VAD-FMK) and an antioxidant (N-acetyl-l-cysteine). In vivo, ß-lapachone and its 3-iodine derivatives significantly reduced tumor burden and did not alter any of the biochemical, hematological, or histological parameters of the animals. Overall, ß-lapachone and its 3-iodine derivatives showed promising cytotoxic activity due to their ability to induce cell cycle arrest at G2/M phase and promote caspase- and ROS-mediated apoptosis. In addition, ß-lapachone and its 3-iodine derivatives were able to suppress tumor growth in vivo, indicating that these compounds may be new antitumor drug candidates.

Anti-liver cancer activity in vitro and in vivo induced by 2-pyridyl 2,3-thiazole derivatives.

A total of 24 hybrid compounds containing pyridyl and 1,3-thiazole moieties were screened against HL-60 (leukemia), MCF-7 (breast adenocarcinoma), HepG2 (hepatocellular carcinoma), NCI-H292 (lung carcinoma) human tumor cell lines and non-tumor cells (PBMC, human peripheral blood mononuclear cells). Most of them were highly potent in at least one cell line tested (IC50≤3µM), being HL-60 the most sensitive and HepG2 the most resistant cell line. Among them, TAP-07 and TP-07 presented cytotoxic activity in all tumor cell lines, including HepG2 (IC50 2.2 and 5.6µM, respectively) without antiproliferative effects to normal cells (PBMC) (IC50>30µM), making TAP-07 and TP-07, the compounds with the most favorable selectivity index. TAP-07 and TP-07 induced apoptosis in HepG2 cells and presented in vivo antitumor activity in hepatocellular xenograft cancer model in C.B-17 severe combined immunodeficient mice. Systemic toxicological verified by biochemical and histopathological techniques reveled no major signs of toxicity after treatment with TAP-07 and TP-07. Together the results indicated the anti-liver cancer activity of 2-pyridyl 2,3-thiazole derivatives.

Glypican-3 distinguishes aggressive from non-aggressive odontogenic tumors: a preliminary study.

BACKGROUND: Glypican-3 is a cell surface proteoglycan that is found in embrionary tissues, and there are no studies investigating this protein in odontogenic tumor. Thus, the aim of this study was to investigate glypican-3 in a series of aggressive and non-aggressive odontogenic tumors. METHODS: Fifty-nine cases of tumors were divided into aggressive odontogenic tumors (20 solid ameloblastomas, four unicystic ameloblastoma, 28 KOTs including five associated with Gorlin-Goltz syndrome) and non-aggressive odontogenic tumors (five adenomatoid odontogenic tumors and two calcifying cystic odontogenic tumors) and analyzed for glypican-3 using immunohistochemistry. RESULTS: Glypican-3 was observed in seven solid ameloblastoma and eighteen keratocystic odontogenic tumors including three of the five syndromic cases, but there was no significant difference between syndromic and sporadic cases (P > 0.05; Fisher's exact Test). All cases of unicystic ameloblastoma (n = 4), adenomatoid odontogenic tumor (n = 5), and calcifying cystic odontogenic tumor (n = 2) were negative. CONCLUSIONS: This provided insights into the presence of glypican-3 in odontogenic tumors. This protein distinguished aggressive from non-aggressive odontogenic tumors.

Macrophages and endothelial cells orchestrate tumor-associated angiogenesis in oral cancer via hedgehog pathway activation.

The present study aimed to evaluate the role of Hedgehog (Hh) molecule expression in association with the clinical aspects of oral squamous cell carcinoma (OSCC), as well as angiogenesis and CD163+ macrophages. Twenty-eight cases of OSCC, nine cases of tumor-free resection margins (TM), and four cases of non-neoplastic oral mucosa (NNM) were submitted to immunohistochemistry to detect proteins Sonic Hedgehog (SHH), Indian Hedgehog (IHH), GLI1, CD163, and CD105. Protein colocalization with respect to SHH/CD163, IHH/CD163, GLI1/CD163, and GLI1/CD105 was assessed by immunohistochemical double staining. In tumor parenchyma, SHH and IHH were present in the cytoplasm of neoplastic cells, while GLI1 was observed in cytoplasm and nucleus. Endothelial cells were found to express SHH, IHH, and GLI1 within CD105+ vessels, and a positive correlation between infiltrating macrophage density (IMD) and microvascular density (MVD) was observed in cases of OSCC and TM. When compared to TM and NNM, the OSCC cases demonstrated higher immunoreactivity for SHH (p = 0.01), IHH (p = 0.39), GLI1 (p = 0.03), IMD (p = 0.0002), and MVD (p = 0.0002). Our results suggest the participation of the Hh pathway in OSCC by way of autocrine and paracrine signaling, in addition to the participation of both SHH and IHH ligands. Endothelial cells were also found to exhibit positivity with respect to Hh pathway components and we surmise that these molecules may play a role in tumor angiogenesis. CD163+ macrophages were also observed to express IHH, a ligand of this pathway, in addition to being associated with tumor neovascularization.

Enhanced Expression of Hedgehog Pathway Proteins in Oral Epithelial Dysplasia.

The aim of this study was to characterize the profile of the proteins involved in the Hedgehog signaling pathway to aid in the understanding of the pathogenesis of oral epithelial dysplasia (OED). The proteins SHH, PTCH1, HHIP, SUFU, GLI1, and cyclin D1 were evaluated by immunohistochemistry in 25 cases of OED, 4 of non-neoplasic oral mucosa, 8 of inflammatory fibrous hyperplasia and 5 of hyperkeratosis. SHH proteins were predominant in OED cases. Although PTCH1 protein was observed in all cases, this molecule was more highly expressed in OED. The inhibitor protein SUFU was present in OED and HHIP protein was overexpressed in OED. GLI1 proteins were predominantly found in the nuclei of epithelial cells in OED. Basal and suprabasal cells in the epithelial lining were positive for cyclin D1 only in OED. In conclusion, comparative analysis of the proteins involved in the Hedgehog pathway suggests that enhanced expression of these proteins can play an important role in the biological behavior of OED.

Association between radiographic features and cell proliferation in keratocystic odontogenic tumor / Asociación entre las características radiográficas y la proliferación celular en el tumor odontogénico queratoquístico

The radiographic features of an intraosseous lesion are usually associated with the biological behavior of the tumor. In view of the fact that the growth and behavior of keratocystic odontogenic tumors (KCOT) is mainly associated with the proliferation of the cystic epithelium, the objective of the present study was to evaluate the relationship between cell proliferation markers and radiographic features of this tumor. Thirty-seven radiographs of KCOT obtained from 30 patients were scanned and evaluated on a monitor. Sections were submitted to immunohistochemistry for Ki-67, p63, and p53 proteins on an EnVision system. Thirty-one KCOTs were observed in the posterior of the mandible, and the unilocular aspect was predominant (n= 26). Nineteen KCOTs distorted the mandibular canal and 11 displaced teeth. Satellite cysts were associated with a multilocular aspect (P= 0.016). p53 was in KCOTS with diffuse margins (p=0.049), p63 with NBCCS (p=0.049) KOT and higher KI-67 positive cells was observed in KCOTs presenting distortion of the mandibular canal (p=0.042). The distribution of Ki-67, p63, and p53 positive cells was similar between KCOTs with uni- and multilocular aspects. The results of the present study suggest that cell proliferation in KCOT contributes to the radiographic features of this tumor.

Las características radiográficas de una lesión intraósea se asocian generalmente con el comportamiento biológico del tumor. Debido a esto, el crecimiento y comportamiento de los tumores odontogénicos queratoquísticos se asocian principalmente con la proliferación del epitelio quístico. El objetivo del estudio fue evaluar la relación entre los marcadores de proliferación celular y las características radiológicas de este tumor. Se escanearon y evaluaron 37 radiografías de tumores odontogénicos queratoquísticos obtenidos de 30 pacientes y las secciones de sus biopsias fueron sometidas a evaluación inmunohistoquímica para las proteíneas Ki-67, p63 y p53 en un sistema Envision. Se observaron 31 tumores odontogénicos queratoquísticos en el área posterior de la mandíbula, con predominio del aspecto unilocular (n= 26). Diecinueve tumores odontogénicos queratoquísticos distorsionaron el canal mandibular y se observaron 11 dientes desplazados. Los quistes satélites se asociaron con el aspecto multilocular (P= 0,016). La distribución de células positivas para Ki-67, p63 y p53 fue similar entre tumores odontogénicos queratoquísticos con aspectos uniformes y multiloculares, y no estaban relacionadod con la distorsión del canal mandibular (P>0,05) o con el desplazamiento del diente (P>0,05). Los resultados del presente estudio sugieren que la proliferación celular en tumores odontogénicos queratoquísticos contribuye a las características radiográficas de este tumor.

Estudo do perfil de expressão de componentes da via de sinalização sonic hedgehog em displasia epitelial oral / Study of the expression profile of components of sonic hedgehog signaling pathway in oral epithelial dysplasia

A displasia epitelial oral (DEO) é um aspecto histológico descrito em lesões potencialmente malignas, cujos mecanismos relacionados a patogênese e potencial de transformação são pouco conhecidos. Sabendo-se que a via de sinalização Sonic Hedgehog(SHH)tem participação no desenvolvimento do carcinoma escamocelular de boca(CEB) e que as proteínas relacionadas a esta via de sinalização estão envolvidas nos mecanismos biológicos relacionados a iniciação e progressão de tumores humanos. O objetivo deste trabalho foi estudar a expressão das proteínas da via de sinalização SHH em DEO, a fim de contribuir para o conhecimento do perfil biológico desta lesão...

Oral epithelial dysplasia (OED) is a histological aspect described in premalignant lesions and themechanisms related to the pathogenesis and malignant progression are poorly understood. It is knownthat Sonic Hedgehog (SHH) signaling pathway participates in the development of oral squamous cellcarcinoma, and proteins related to this cascade are involved in biological mechanisms related to theinitiation and progression of human tumors. The aim of this study was to investigate SHH signalingmolecules in OED, in order to contribute to the knowledge of the biological profile of this lesion...

Obesidade, um possível indicador de risco para a doença periodontal? / Obesity, a possible risk indicator for periodontal disease?

A prevalência da obesidade vem aumentando nas últimas décadas, representando uma preocupação para a saúde pública. Essa patologia é um importante fator de risco para o desenvolvimento de várias doenças sistêmicas,como diabetes mellitus tipo II, hiperlipidemia, hipertensão, doenças cardiovasculares e colelitíase.Estudos epidemiológicos recentes demonstraram uma possível relação entre a obesidade e a periodontite,uma patologia infecto-inflamatória do tecido gengival e do periodonto de sustentação, cujo fator etiológico primário é a presença do biofilme bacteriano nos dentes. A associação entre a periodontite e a obesidade está diretamente relacionada ao processo inflamatório, pois mediadores pró-inflamatórios são secretados pelo tecido adiposo, o que faz com que estejam presentes em maior quantidade em pacientes obesos, podendo, por conseguinte, levar a um estado hiperinflamatório, aumentando o risco ou a progressão das doenças periodontais. Diante disso, o objetivo deste trabalho é apresentar e discutir, por meio de uma revisão de literatura, as evidências científicas e os mecanismos biológicos que mostram a obesidade como um possível indicador de risco para a periodontite.

The prevalence of obesity has increased in recent decades, representing a concern for public health. Thisdisease is an important risk factor for the development of several systemic diseases such as diabetes mellitustype II, hyperlipidemia, hypertension, cardiovascular disease and cholelithiasis. Recent epidemiologicalstudies have shown a possible link between obesity and periodontitis, which is an infectious inflammatorydisease of the gingival tissue and periodontal support, whose primary etiological factor is thepresence of biofilm on the teeth. The association between periodontitis and obesity is directly related tothe inflammatory process, as pro-inflammatory mediators are secreted by adipose tissue, which are presentin greater quantities in obese patients and may therefore lead to a hypertensive state inflammation,increasing the risk or progression of periodontal diseases. Thus, the objective was to provide, through aliterature review, the scientific evidence and biological mechanisms that show obesity as a possible riskindicator for periodontitis.

Estudo da obesidade como indicador de risco para a doença periodontal / Study of obesity as a risk indicator for periodontal disease

A prevalência da obesidade vem aumentando nas últimas décadas, representando uma preocupação para a saúde pública, pois esta condição é um importante fator de risco para o desenvolvimento de várias doenças sistêmicas. Estudos epidemiológicos recentes demonstraram uma possível relação entre a obesidade e a doença periodontal. Esta associação está diretamente relacionada ao processo imuno-inflamatório, pois mediadores inflamatórios são secretados pelo tecido adiposo, o que faz com que estejam presentes em maior quantidade em pacientes obesos, podendo, por conseguinte levar a um estado hiperinflamatório, aumentando o risco ou a progressão da doença periodontal. A partir disso, o objetivo deste trabalho foi verificar, por meio de um estudo transversal, a possível associação entre a obesidade e a doença periodontal. A amostra consistiu de 100 pacientes não fumantes, sistemicamente saudáveis, que não receberam tratamento periodontal nos últimos 6 meses ou usado antibiótico e/ou anti-inflamatório nos últimos 3 meses. O exame clínico periodontal completo, o índice de massa corporal (IMC) e a medida da circunferência abdominal foram realizados. A periodontite foi encontrada em 79% dos indivíduos avaliados, o sobrepeso em 34% e a obesidade em 23% dos casos. Já a circunferência abdominal encontrada foi > 88 cm em 54,79% das mulheres avaliadas e > 102 cm em 25,93% dos homens. Neste estudo, não houve associação entre a periodontite e a obesidade, porém, estas foram prevalentes, tornando-se uma preocupação para a saúde pública. A partir disso, medidas preventivas são necessárias, visando à prevenção e promoção de saúde para a população estudada

The prevalence of obesity has increased in recent decades, representing a public health concern because this condition is an important risk factor for the development of several systemic diseases. Recent epidemiological studies have shown a possible link between obesity and periodontal disease. This association is directly related to the immunoinflammatory process, because inflammatory mediators are secreted by adipose tissue, which causes that are present in greater quantities in obese patients and may therefore lead to a state hiperinflamatório, increasing the risk or progression periodontal disease. From this, the objective was to verify,through a cross-sectional study of the possible association between obesity and periodontal disease. The sample consisted of 100 non-smoking patients, systemically healthy, who had not received periodontal treatment with in the last 6 months or used antibiotics and / or inflammatory in the last three months. The clinical examination is complete, the body mass index (BMI) and waist circumference were performed. Periodontitis was found in 79% of the individuals, overweightand obesity in 34% to 23% of cases. Already found waist circumference was > 88 cm in 54,79% the women studiedand > 102 cm in 25, 93% of me. In this study, no association between periodontitis and obesity, but these were prevalent, becoming a concern for public health. From this, preventive measures are necessary, aiming at prevention and health promotion for this population.