Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan

Abstract

Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5 x 10(5) cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-3H]--lysine labelled purified chick--embryo aorta elastin substrate. After incubation for 10 h at 37 degrees C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84 +/- 1.15 mg/ml) and lysyl oxidase activity (3558.6 +/- 345.5 cpm/10(6) cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35 +/- 0.96 mg/ml and 2436.0 +/- 352.6 cpm/10(6) cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.

Therapeutic Interventions in Oral Submucous Fibrosis: An Experimental and Clinical Study

“…This is a copper dependent enzyme [19] and plays a key role in collagen synthesis. The possible role of copper as a mediator of fibrosis is supported by the demonstration of up regulation of this enzyme in OSF biopsies [30] and in OSF fibroblasts compared to normal fibroblasts grown in culture [21]. We also found a similar effect in our case and the effect was more prominent when these cells were exposed to arecoline 10 lM, 24 h. b-aminopropionitril (BAPN, 100 lM), an irreversible LOX inhibitor, was found to complete reversal of LOX activity as measured by ELISA and western blot analysis (Fig.…”

“…Current evidence implicates collagen-related genes in the susceptibility and pathogenesis of OSF [9]. The activity of lysyl oxidase (LYOXase), an extracellular enzyme which stabilizes the collagen and prevents degradation of collagenase in oral mucosa, was found to increase in fibroblasts cultured from OSF patients [21].…”

In vivo autofluorescence spectroscopy of oral premalignant and malignant lesions: Distortion of fluorescence intensity by submucous fibrosis

“…These atrophic epithelial or cancer tissues might contain less amount of NADH and thus had a lower 460-nm emission peak than the corresponding lesions in non-OSF patients. In Taiwan, oral premalignant and malignant lesions are commonly found in OSF patients and AQ-chewing habit prevails in patients with oral premalignant and malignant lesions [22]. As AQ-chewing habit may cause obscure to different degrees of fibrosis in the oral mucosa, autofluorescence spectra measured from premalignant, malignant and their surrounding normal tissues in AQ-chewers or OSF patients may be distorted due to the presence of underlying tissue fibrosis.…”

Estimation of Serum Copper and Zinc Levels in Oral Submucous Fibrosis: An Atomic Absorption Spectroscopic Study

“…6,14,15 The increase in copper may also be attributed to the increased production of copper containing ceruloplasmin by liver as an anti-inflammatory response or due to decrease in catabolism of serum ceruloplasmin. 16 In our study, the mean serum zinc level among 30 OSMF patients was found to be lower than the controls.…”