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.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization.

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

Effects of IGF-1 on Proliferation, Angiogenesis, Tumor Stem Cell Populations and Activation of AKT and Hedgehog Pathways in Oral Squamous Cell Carcinoma.

(1) Background: Activation of the PI3K-AKT pathway controls most hallmarks of cancer, and the hedgehog (HH) pathway has been associated with oral squamous cell carcinoma (OSCC) development and progression. We hypothesized that fibroblast-derived insulin-like growth factor-1 (IGF-1) acts in oral squamous cell carcinoma (OSCC) cells, leading to the non-canonical activation of the HH pathway, maintaining AKT activity and promoting tumor aggressiveness. (2) Methods: Primary fibroblasts (MF1) were genetically engineered for IGF-1 overexpression (MF1-IGF1) and CRISPR/Cas9-mediated IGF1R silencing was performed in SCC-4 cells. SCC-4 cells were co-cultured with fibroblasts or incubated with fibroblast conditioned medium (CM) or rIGF-1 for functional assays and the evaluation of AKT and HH pathways. (3) Results: Gene expression analysis confirmed IGF-1 overexpression in MF1-IGF1 and the absence of IGF-1 expression in SCC-4, while elevated IGF1R expression was detected. IGF1R silencing was associated with decreased survival of SCC-4 cells. Ihh was expressed in both MF1 and MF1-IGF1, and increased levels of GLI1 mRNA were observed in SCC-4 after stimulation with CM-MF1. Activation of both PI3K-AKT and the HH pathway (GLI1, Ihh and SMO) were identified in SCC-4 cells cultured in the presence of MF1-IGF1-CM. rIGF-1 promoted tumor cell proliferation, migration, invasion and tumorsphere formation, whereas CM-MF1 significantly stimulated angiogenesis. (4) Conclusions: IGF-1 exerts pro-tumorigenic effects by stimulating SCC-4 cell proliferation, migration, invasion and stemness. AKT and HH pathways were activated by IGF-1 in SCC-4, reinforcing its influence on the regulation of these signaling pathways.

Central giant cell granuloma: A clinicopathological and immunohistochemical study of macrophages, blood vessels, lymphatic vessels and regulatory proteins.

OBJECTIVE: This study seeks to investigate immunohistochemical parameters that could distinguish non-aggressive Central giant cell granuloma (CGCG) from aggressive CGCG, two groups of lesions which differ in their clinical and radiographic features and prognosis. MATERIAL AND METHODS: 12 cases of non-aggressive CGCG and 11 cases of aggressive CGCG were investigated and associated the immunohistochemical expression of macrophages (CD68 and CD163), blood vessels (CD34 and CD105), lymphatic vessels (D2-40) and regulator proteins (p63 and Ki-67). Clinical and radiographic features were also studied. RESULTS: Associations between all proteins in non-aggressive and aggressive CGCG were not significant (p > 0.05). With respect to non-aggressive CGCG, there were no significant correlations, while in aggressive CGCG there was a significant positive correlation between CD68 and CD163 (p = 0.031), between CD34 and D2-40 proteins (p = 0.04), whereas a significant negative correlation was observed between CD105 and CD68 (p = 0.040). However, regardless of aggressiveness of CGCG, there was a significant positive correlation between CD68 and CD163 (p = 0,04). Among the clinical and immunohistochemical aspects, only the symptomatology was a significant risk factor for the occurrence of aggressive CGCG (OR = 12.00/p = 0.016). CONCLUSION: Macrophages and angiogenesis contribute to their maintenance and development of CGCG. In addition, immunohistochemistry used here was not able to differentiate their aggressiveness. However, symptomatology was proved to be a risk factor for the occurrence of aggressive CGCG. It is possible that clinical features, particularly symptomatology, represent the most appropriate parameter to attempt to distinguish GCCG.

Trifid nasopalatine canal: case report of a rare anatomical variation and its surgical implications / Conducto nasopalatino Trifid: presentación de caso de una variación anatómica rara y sus implicaciones quirúrgicas

The nasopalatine canal is a long slender structure present in the midline of the anterior maxilla that connects the palate to the floor of the nasal cavity. The nasopalatine canal contains the nasopalatine nerve, the terminal branch of the nasopalatine artery, fibrous connective tissue, adipose tissue, and minor salivary glands. The purpose of this article was to report a case of a trifid nasopalatine canal detected by cone beam computed tomography prior to dental implant placement. A 47-year-old female patient was submitted to cone beam computed tomography. Axial and sagittal sections revealed a trifurcation of the nasopalatine canal. Each canal was separated from the other by bony septa and extended independently from the floor of the nasal cavity to the incisive foramen in the remnant of the alveolar process in the anterior region of the maxilla. Cone beam computed tomography has permitted better visualization of the details and anatomical variations of the nasopalatine canal. Detailed knowledge of variations in the shape, number and size of the nasopalatine canal is fundamental for surgical procedures, such as local anesthesia in the anterior maxillary region and placement of dental implants, in order to prevent damage to important arteries and nerves(AU)

El canal nasopalatino es una larga estructura delgada presente en la línea media del maxilar anterior que conecta el palato al suelo de la cavidad nasal. El canal nasopalatino contiene el nervio nasopalatino, la rama terminal de la arteria nasopalatina, el tejido conectivo fibroso, el tejido adiposo y las glándulas salivales menores. El propósito de este artículo es presentar el caso de un canal nasopalatino trifid detectado a través de tomografía computarizada de haz cónico anterior a la colocación de implantes dentales, en una paciente de femenino 47 años de edad. Secciones axiales y sagitales revelaron la trifurcación del canal nasopalatino. Cada canal se apartó del otro por tabiques ósea y extendida independientemente del suelo de la cavidad nasal para el agujero incisivo en el remanente del proceso alveolar en la región anterior del maxilar. La tomografía computarizada de haz cónico ha permitido una mejor visualización de los detalles y variaciones anatómicas del canal nasopalatino. El conocimiento detallado de las variaciones en su forma, el número y el tamaño del canal nasopalatino es fundamental para los procedimientos cirúrgicos, así como la anestesia local en la región anterior del maxilar superior y la colocación de los implantes dentales, con el fin de prevenir el daño a las arterias y a los nervios importantes(AU)

Elevated VEGFA mRNA levels in oral squamous cell carcinomas and tumor margins: a preliminary study.

BACKGROUND: New blood vessel formation events are essential for tumor clonal expansion and progression. Despite the importance of vascular endothelial growth factor A (VEGFA) for tumor angiogenesis, few studies have evaluated the expression profile of this gene in oral squamous cell carcinoma (OSCC) and tumor margins (TM). This study aimed to evaluate the expression of the VEGFA gene and its protein in OSCC and TM. METHODS: Gene expression was evaluated in cryopreserved samples of OSCCs (n = 44), TM (n = 7), and normal mucosa from healthy patients (n = 3; NM). Quantitative PCRs were performed using the TaqMan system for the VEGFA gene, and gene expression was determined using the 2(-∆∆CQ) method. For immunohistochemical evaluation, 27 OSCC samples were embedded in a tissue microarray (TMA) block and reactions were performed using the Advance system. RESULTS: VEGFA transcript levels were 1.7-fold higher in OSCC than in NM samples. VEGFA mRNA was overexpressed in TM samples compared to NM (3.4-fold) and OSCC (2.0-fold) samples. VEGFA transcript level was overexpressed in T3-T4 tumors, metastatic lymph nodes, and stage III-IV OSCCs. Immunoreactivity of the VEGFA protein was detected in the cytoplasm of parenchymal and stromal cells, mainly in endothelial cells and fibroblasts. CONCLUSION: Our results show that VEGFA was overexpressed in aggressive OSCCs and that VEGFA expression may be an important prognostic factor in this type of cancer. Finally, tumor margins may be involved in the production of angiogenic molecules.

Transcriptional profiles of SHH pathway genes in keratocystic odontogenic tumor and ameloblastoma.

BACKGROUND: Sonic hedgehog (SHH) pathway activation has been identified as a key factor in the development of many types of tumors, including odontogenic tumors. Our study examined the expression of genes in the SHH pathway to characterize their roles in the pathogenesis of keratocystic odontogenic tumors (KOT) and ameloblastomas (AB). METHODS: We quantified the expression of SHH, SMO, PTCH1, SUFU, GLI1, CCND1, and BCL2 genes by qPCR in a total of 23 KOT, 11 AB, and three non-neoplastic oral mucosa (NNM). We also measured the expression of proteins related to this pathway (CCND1 and BCL2) by immunohistochemistry. RESULTS: We observed overexpression of SMO, PTCH1, GLI1, and CCND1 genes in both KOT (23/23) and AB (11/11). However, we did not detect expression of the SHH gene in 21/23 KOT and 10/11 AB tumors. Low levels of the SUFU gene were expressed in KOT (P = 0.0199) and AB (P = 0.0127) relative to the NNM. Recurrent KOT exhibited high levels of SMO (P = 0.035), PTCH1 (P = 0.048), CCND1 (P = 0.048), and BCL2 (P = 0.045) transcripts. Using immunolabeling of CCND1, we observed no statistical difference between primary and recurrent KOT (P = 0.8815), sporadic and NBCCS-KOT (P = 0.7688), and unicystic and solid AB (P = 0.7521). CONCLUSIONS: Overexpression of upstream (PTCH1 and SMO) and downstream (GLI1, CCND1 and BCL2) genes in the SHH pathway leads to the constitutive activation of this pathway in KOT and AB and may suggest a mechanism for the development of these types of tumors.