Abstract
Background: The lysyl oxidase family of enzymes consists of five members, namely lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) 1-4. They are secreted enzymes whose function is to stabilise the extracellular matrix via crosslinking collagens and elastins. The enzymes are mostly known for their extracellular function but little is known about their intracellular function. Few studies have investigated LOX expression in the oral and maxillofacial region and those that exist are largely concerned with oral submucous fibrosis and oral squamous cell carcinoma. With regard to odontogenic lesions one study exists which showed increased expression of LOXL4 in the stromal tissue of odontogenic keratocyst (OKC).
Objective: To determine the expression of LOX family proteins and genes in locally aggressive odontogenic lesions (ameloblastoma and OKC) in comparison with non-aggressive odontogenic lesions (dentigerous cyst (DC) and hyperplastic dental follicle (DF)) using immunohistochemistry (IHC) and quantitative reverse transcriptase real-time polymerase chain reaction (qRT2-PCR).
Method: For IHC, formalin-fixed paraffin-embedded (FFPE) tissue samples of ameloblastoma (n = 10), OKC (n = 15), DC (n = 6) and DF (n = 9) were used with antibodies against LOX and LOXL1-4. Positive and negative controls were used for validation. Qualitative assessment of the pattern and distribution of staining was undertaken at varying magnifications. Automated quantitative assessment of digitised IHC images was performed using Fiji Software (Image J 1.51K). Specifically, the extent of positive reaction and intensity of staining were examined in three representative areas of the epithelium and connective tissue in each specimen at 400x magnification. One way ANOVA tests were performed using GraphPad Prism software (La Jolla California USA), and P values of <0.05 were considered to represent a statistically significant difference between the groups. For qRT2-PCR, RNA samples were isolated from FFPE tissue sections of ameloblastoma, OKC and DC, which were subsequently reverse transcribed into cDNA. The cDNA samples were preamplified prior to performing PCR reactions. A reference gene was used to normalise the results. The expression level of each gene and fold regulation (FR) between disease groups was determined using the method. Statistical significance was denoted when FR>2 and p<0.05 for each analysis.
Result: The LOX family proteins and genes showed differential patterns of expression in each lesion examined. Significant reduction of LOXL3 was observed in ameloblastoma at both protein and gene levels. LOXL4 protein was overexpressed in the epithelium, but underexpressed in the connective tissue of ameloblastoma and OKC. The expression of LOX family genes and proteins in DC showed a significant variation compared with ameloblastoma and OKC, whereas the protein expression patterns were similar between ameloblastoma and DF.
Conclusion: 1) LOX family expression was different in ‘aggressive’ odontogenic lesions compared with ‘non-aggressive’ odontogenic lesions, dentigerous cyst in particular. 2) Variable LOX family expression between ameloblastoma and OKC may reflect their pathogenesis and biological behaviour. 3) The similarities of LOX family expression observed in ameloblastoma and hyperplastic dental follicle may reflect the embryonic dedifferentiation of ameloblastoma. 4) Dentigerous cyst serves as a proper ‘control’ tissue as opposed to hyperplastic dental follicle with regard to study of the LOX family.