Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

BACKGROUND: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. METHODS: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. RESULTS: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. CONCLUSION: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

Self-assembly peptide P11-4 induces mineralization and cell-migration of odontoblast-like cells.

OBJECTIVES: Self-assembling peptide P11-4 is amphiphilic and pH-triggered, effective on repairing early enamel carious lesions and dentin remineralization. However, P11-4 effects on dentin biomineralization and repair ability remain unexplored. Thus, cytocompatibility and effectiveness of P11-4 on inducing mineralization and migration of odontoblast-like cells (MDPC-23) were investigated. METHODS: MDPC-23 were seeded in contact with P11-4 (0.5 and 1 µg/ml), Dentin Matrix Protein 1 (DMP1 0.5 and 1 µg/ml) or Calcium hydroxide (Ca(OH)2 100 µg/ml) solutions. Cell viability was verified using MTT (n = 6/group). Mineral deposition was tested using Alizarin Red (n = 4/group). Cell migration was assessed by light microscopy (n = 2/group). MTT and Alizarin Red data were compared using Kruskal-Wallis and Mann-Whitney (α=0.01). RESULTS: P11-4 (0.5 and 1 µg/ml) and DMP1 (0.5 and 1 µg/ml) resulted the highest cell viability; Ca(OH)2 presented the lowest. 1 µg/ml DMP1 and 1 µg/ml P11-4 promoted the highest mineral deposition. Ca(OH)2 presented lower values of mineral deposits than DMP1 1 µg/ml (p < 0.01), but similar to P11-4 1 µg/ml. P11-4 and DMP1 at 0.5 µg/ml induced lesser mineral precipitation than P11-4 and DMP1 at 1 µg/ml (p < 0.01), with no difference to Ca(OH)2. All materials stimulated cell migration, however, lower concentrations of DMP1 and P11-4 demonstrated a higher migration potential. CONCLUSION: P11-4 did not affect cell viability, induces mineral deposition and MDPC-23 migration like DMP1. CLINICAL SIGNIFICANCE: Self-assembling peptide P11-4 does not affect the cell viability and induces mineral deposition comparable to native protein involved in biomineralization. Combined with its ability to bind type I collagen, P11-4 is a promising bioinspired molecule that provides native-tissue conditions and foster further studies on its ability to form dentin bridges in pulp-capping strategies.

Combined therapy with cisplatin and 5-AZA-2CdR modifies methylation and expression of DNA repair genes in oral squamous cell carcinoma.

The methylation and expression of DNA repair system genes has been studied in several tumor types. These genes have been associated with resistance to chemotherapy treatments by epigenetic regulation. Studies have yet to show the effects of combined therapy using an epigenetic drug (5-aza-2CdR) and cisplatin (CDDP) on DNA repair genes in oral squamous cell carcinoma (OSCC). This study proposed to investigate the effects of CDDP in combination with 5-aza-2CdR on the methylation of MGMT and MLH1 genes in oral cancer cells. Oral squamous cell carcinoma cell lineages (SCC-9, SCC-15, and SCC-25) were submitted to 72 hours of treatment: 0.1 µM CDDP (or 4.44 µM SCC-9), 0.1 µM and 0.3 µM 5-aza-2CdR (or 1 µM and 3 µM SCC-9), and the drugs in combination. Cell viability was assessed by MTT, DNA methylation of MGMT and MLH1 genes by Methylation Sensitivity High-Resolution Melting (MS-HRM), and the relative expression of the genes by RT-qPCR. The results show that all treatments reduced cell viability; however, in SCC-15 and SCC-9 (IC50 value), 5-aza-2CdR promotes cell sensitization to cytotoxic effect of cisplatin. The MGMT promoter region was 100% demethylated in the SCC-15 and SCC-25 cells but partially (50%) methylated in SCC-9 before drug treatment. Treatment with IC50 CDDP value kept the methylation status and decreased MGMT expression in SCC-9; MGMT gene in SCC-15 and SCC-25 cells became downregulated after treatment with 5-aza-2CdR. MLH1 was demethylated, but the treatments with low-doses and combined drugs decreased the expression in SCC-9 and SCC-25; however high doses of 5-aza-2CdR and drug combination with IC50 value CDDP increased expression of MLH1 in SCC-9. The data presented suggest that epigenetic drugs associated with chemotherapy have clinical translational potential as a therapy strategy to avoid or reverse cancer resistance, requiring further investigation.

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

Cell culture of the normal human mammary gland cultivated in monolayer - A mini systematic review.

The mammary glands are constituted of different cell types. For example, the epithelial cells appear as the target in many studies since they produce and secrete milk during lactation and are the origin of many human breast cancers. Mammary gland biology is characterized by dynamic tissue growth, function and regression phases, which are understood mainly due to tissue culture studies. Cell culture is probably one of the most used in vitro scientific models, and the most common research model is still the two-dimensional (2D) culture system. Different approaches and conditions have been tested and used to improve the isolation, growth, yield and maintenance of viability of mammary gland cells. Therefore, our study aimed to explore and summarize the cell culture techniques with normal human mammary gland cells cultured in a monolayer. A search strategy was conducted using the electronic databases 'PubMed', 'Scopus' and 'Virtual Health Library'. The search was carried out using the keywords 'cell culture' and 'mammary gland' and 'human'. The main search was carried out by two authors between July and August 2021. In addition, we performed a review matrix elaborated in a spreadsheet to organize and systematize information about each article for inclusion. A total of 11 studies were included in the review and have conducted qualitative analyses on them. Although studies of these cells have been reported since the 1970 s, most found are from the last decade and are largely carried out in the USA. In addition, it was possible to verify the Human Mammary Epithelial Cells (HMEC) primary culture obtained from breast surgery as the main cell type studied. These cells are cultivated in Dulbecco's Modified Eagle Medium (DMEM) and M87A medium with diverse supplements. Finally, there was a diversity in the use of dissociation reagents and a lack of information about cryopreservation. We have observed detailed methodological information about these study models, which would propose further investigations.

Addition of hydrogen peroxide to methylene blue conjugated to ß-cyclodextrin in photodynamic antimicrobial chemotherapy in S. mutans biofilm.

OBJECTIVE: This study evaluated the effect of hydrogen peroxide addition on ß-cyclodextrin-conjugated methylene blue in antimicrobial photodynamic therapy(a-PDT) in S. mutans biofilm model using laser or light emitting diode (LED) (λ = 660 nm). METHODS: A preliminary assay was performed to evaluate the cytotoxicity of hydrogen peroxide in oral fibroblasts by the colorimetric method (MTT). Afterwards, groups were divided into (n = 3, in triplicate): C (negative control), CX - chlorhexidine 0.2% (positive control), P (methylene blue/ß-cyclodextrin), H (Hydrogen Peroxide at 40 µM), PH, L (Laser), LP, LH (Laser+Hydrogen Peroxide), LPH, LED, LEDP, LEDH, and LEDPH. The biofilm was formed in 24 h with BHI + 1% sucrose (w/v). Light irradiations were conducted with laser, 9 J, 323 J/cm2, 113 s or with LED, 8.1 J, 8.1 J/cm2 for 90 s. Microbial reduction was evaluated by counting the viable microorganisms of the biofilm after the respective treatments, in a selective culture medium, and laser confocal microscopy evaluation. RESULTS: LP, LH, LPH, LEDP, LEDH, and LEDPH groups statistically reduced the counts of S.mutans compared with the C group and the log reductions were of 1.87, 1.94, 2.19, 0.91, 0.92, and 1.33, respectively; the addition of hydrogen peroxide did not potentiate the microbial reductions (LPH and LEDPH) compared with the LP and LEDP groups. CONCLUSION: The association of hydrogen peroxide with the conjugated ß-cyclodextrin nanoparticle as photosensitizer did not result in an enhanced effect of a-PDT; hydrogen peroxide behaved as a photosensitizer, since it reduced the number of S. mutans when associated with laser light.

Parathyroid hormone (1-34) modulates odontoblast proliferation and apoptosis via PKA and PKC-dependent pathways.

Parathyroid hormone (PTH) plays a key role in the development and homeostasis of mineralized tissues such as bone and dentine. We have reported that PTH (1-34) administration can increase dentine formation in mice and that this hormone modulates in vitro mineralization of odontoblast-like cells. The purpose of the present study was to investigate whether PTH (1-34) participates in the proliferative and apoptotic signaling of odontoblast-like cells (MDPC23). MDPC23 cells were exposed to 50 ng/ml hPTH (1-34) or vehicle for 1 (P1), 24 (P24), or 48 (P48) hours, and the cell proliferation, apoptosis, and cell number were evaluated. To examine whether changes in the proliferative and apoptotic signaling in response to PTH involve protein kinases A (PKA) and/or C (PKC), MDPC23 cells were exposed to PTH with or without PKC or PKA signaling pathway inhibitors. Overall, the results showed that the PKA pathway acts in response to PTH exposure maintaining levels of cell proliferation, while the PKC pathway is mainly involved for longer exposure to PTH (24 or 48 h), leading to the reduction of cell proliferation and increase of apoptosis. The exposure to PTH reduced the cell number in relation to the control group in a time-dependent manner. In conclusion, PTH modulates odontoblast-like cell proliferative and apoptotic response in a time-dependent manner. Both PKC and PKA pathways participate in PTH-induced modulation in an antagonist mode.

In situ study of the gelatinase activity in demineralized dentin from rat molar teeth.

Matrix metalloproteinases (MMPs) in dentin are believed to participate in various physiological and pathological events in coronal dentin, but their exact source and location is not clear. The purpose of this study was to evaluate the activity of gelatinases in decalcified rat molars crowns by in situ zymography. Hemi-mandibles of five male Wistar rats were fixed in paraformaldehyde, decalcified in EDTA and glycerol solution and embedded in paraffin. Sections from the region of molar teeth were incubated with or without DQ gelatin in 50mM Tris-CaCl2 at 37°C for 2h and observed by means of confocal microscopy. Gelatinolytic activity was observed throughout the coronal dentin with varying intensities in different locations. High gelatinase activity was observed in the dentinal tubules, dentin-enamel junction (DEJ) and predentin, and it was weaker and less uniform in the intertubular dentin. This study shows that the location of gelatinase and relative activity can be detected by means of in situ zymography and confocal microcopy, and this methodology may provide a useful tool in studies on the role of gelatinases in tooth development, maturation and in pathological conditions.

Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes.

OBJECTIVE: The role of epigenetic regulation in inflammatory diseases such as periodontitis is poorly known. The aim of this study was to assess whether Porphyromonas gingivalis lipopolysaccharide (LPS) can modulate gene expression levels of the some enzymes that promote epigenetic events in cultures of the human keratinocytes and gingival fibroblasts. In addition, the same enzymes were evaluated in gingival samples from healthy and periodontitis-affected individuals. MATERIALS AND METHODS: Primary gingival fibroblast and keratinocyte (HaCaT) cultures were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24 h. After this period, cell viability was assessed by MTT test and total RNA extracted to evaluate gene expression levels of the following enzymes by qRT-PCR: DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), histone demethylases Jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX). To evaluate gene expression in healthy and periodontitis-affected individuals, total RNA was extracted from biopsies of gingival tissue from healthy and periodontitis sites, and gene expression of DNMT1, DNAMT3a, JMJD3, and UTX was evaluated by qRT-PCR. RESULTS: No significant differences were found in the gene expression analysis between healthy and periodontitis-affected gingival samples. The results showed that LPS downregulated DNMT1 (p < 0.05), DNMT3a (p < 0.05), and JMJD3 (p < 0.01) gene expression in HaCaT cells, but no modulation was observed in gingival fibroblasts. CONCLUSION: P. gingivalis LPS exposure to human HaCaT keratinocytes downregulates gene expression of the enzymes that promote epigenetic events. CLINICAL RELEVANCE: The advance knowledge about epigenetic modifications caused by periodontopathogens may to possibly led to the development of new periodontal therapies.

Evaluation of the effects of transient or continuous PTH administration to odontoblast-like cells.

Parathyroid hormone participates in the metabolism of mineralized tissue. Its role in the formation of dentine is, as yet, incompletely understood. In the present study we analyzed the effect of transient (1 and 24-h/cycle) or continuous hPTH (1-34) treatment in odontoblast-like cells (MDPC-23) to the following parameters: mineral deposition detected by alizarin red, mRNA expression of the type I collagen (COL1), alkaline phosphatase (ALP), biglycan (BGN), matrix metalloproteinase 2 (MMP-2) and dentine sialophosphoprotein (DSPP) quantified by qRT-PCR. MMP-2 and ALP activities were quantified by zymography and colorimetric assay, respectively. The results showed that compared to Control group: intermittent PTH administration (1 and 24-h/cycle) decreased the mineral deposition and ALP activity. DSPP gene expression was not detected in both control and PTH treated cells. The PTH administration for 24-h/cycle increased the ALP, BGN and COL1 mRNA expression and continuous PTH treatment increased BGN and COL1 mRNA expression. Zymography assays showed that compared to Control group: PTH treatment for 1-h/cycle increased the total MMP-2 secretion and the continuous treatment decreased the secreted levels of MMP-2 active-form. Taken together, the results shown that PTH may regulate the odontoblast-like cells-induced secretion, and potentially this hormone can affect in vivo odontoblasts functions.

Short-term PTH administration increases dentine apposition and microhardness in mice.

OBJECTIVE: The purpose of this study is to investigate the effects of intermittent parathyroid hormone (PTH) administration on the apposition rate and structural features of dentine from mouse incisors. METHODS: Young male A/J Unib mice were treated daily for 6 and 10 days with 40 µg/kg of hPTH 1-34 or a vehicle. Dentine apposition rates measured by fluorescent labels (tetracycline and calcein) and alkaline phosphatase (ALP) plasma levels were evaluated after 6 days of treatment. Knoop microhardness testing and element content measurements in at.% of calcium (Ca), phosphorus (P), oxygen (O), and magnesium (Mg) in the peritubular and intertubular dentine were performed by Energy Dispersive X-ray (EDX) microanalysis via Scanning Electron Microscopy (SEM) after 10 days of treatment. RESULTS: Histometric analysis revealed an increase of 5% in the apposition rate of dentine and 25% in the ALP plasma levels in the PTH treated group. In addition, knoop microhardness testing revealed that the animals treated with PTH had a greater microhardness (11%). EDX microanalysis showed that PTH treatment led to increases in P (23%) and Ca (53%) at.% content, as well as the Ca/P ratio (24%) in peritubular dentine. The chemical composition of intertubular dentine did not vary between the groups. CONCLUSIONS: These findings indicate that intermittent administration of hPTH (1-34) increases apposition and mineralization of the dentine during young mice incisor formation.

Fluoride effect on the secretory-stage enamel organic extracellular matrix of mice.

The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.