Cancer progression is associated with increased expression of basement membrane proteins in three-dimensional in vitro models of human oral cancer

Background

Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front.

Objective

This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial-mesenchymal interactions play on it, by using an in vitro oral cancer progression model.

Material and methods

In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin.

Results

Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium-biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin.

Conclusions

These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial-mesenchymal environment, respectively the existence of fibroblasts.     

Khat induces G1-phase arrest and increased expression of stress-sensitive p53 and p16 proteins in normal human oral keratinocytes and fibroblasts

Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16^INK4A (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21^WAF1/CIP1 (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo , especially in keratinocytes.     

Gut luminal lactate measured by microdialysis mirrors permeability of the intestinal mucosa after ischemia

The aim of the present study was to investigate the influence of a prolonged initial intestinal ischemic insult on transmucosal permeability after a subsequent ischemic event and whether microdialysis of biomarkers released to the gut lumen is able to reflect changes in intestinal permeability. The superior mesenteric artery was cross-clamped for 60 min followed by 4 h of reperfusion in 16 pigs. Nine pigs had a second cross-clamp of 60 min and 3 h of reperfusion, whereas seven pigs were observed for a further 4 h of reperfusion. Intestinal mucosal integrity was assessed by permeability of C-polyethylene glycol (PEG-4000) over the gut mucosa, luminal microdialysis of lactate, glucose and glycerol, and tonometry. During reperfusion, the PEG-4000 amount in venous blood was two times higher after the first than after the second ischemia (area under the curve, 44,780 [13,441-82,723] vs. 22,298 (12,213-49,698] counts min mL(-1), P=0.026 [mean {range}]). There was less lactate detected in the gut lumen after the second ischemia compared with the first (area under the curve, 797 [412-1,700] vs. 1,151 [880-1,969] mmol min L(-1), P=0.02) and a lower maximum concentration (4.8 [2.7-9.4] vs. 8.5 [5.0-14.9] mM, P=0.01). The same pattern was also seen for luminal glycerol and glucose. During the second ischemia, the intestinal mucosal/arterial CO2 gap was identical to the level during the first ischemic episode. A prolonged ischemic insult of the intestine confers protection, for reduced hyperpermeability against further ischemia. Microdialysis of biomarkers mirrors permeability changes associated with this type of protection. Lactate reflects permeability across the intestinal mucosa more precisely than glycerol.     

Disability, pain, psychological factors and physical performance in healthy controls, patients with sub-acute and chronic low back pain: a case-control study.

OBJECTIVE: To compare measures of disability, psychological factors, pain and physical performance in healthy controls, and patients with sub-acute and chronic low back pain. To evaluate the concept of the deconditioning syndrome and to explore factors that may contribute to chronicity. DESIGN: Case-control study. SUBJECTS: Three age- and gender-matched groups were included in the study; healthy controls (n = 45), patients sick-listed 8-12 weeks (n = 46) and patients with chronic low back pain on a waiting list for lumbar instrumented fusion (n = 45). METHODS: Measures of disability, pain, psychological factors, and physical performance were obtained from the 3 groups using validated measures. RESULTS: Gender, age, body weight and height were not significantly different between the groups. Comparable scores were found for self-rated working ability, fear-avoidance beliefs for physical activity and aerobic capacity in the 2 patient groups. Oswestry Disability Index, pain, emotional distress, abdominal and back muscle endurance were significantly different between the 3 groups. Self-efficacy for pain and fear-avoidance beliefs for work was significantly different between the 2 patient groups. CONCLUSION: The results suggest a stepwise deterioration of impairment and disability from healthy controls to patients with chronic low back pain. Most variables distinguished between healthy controls and patients with sub-acute or chronic low back pain. Deconditioning was more related to psychophysical measures of abdominal and back muscle endurance than to cardiovascular fitness. Comparable scores for fear-avoidance and working ability in the 2 patient categories suggest that these factors appear at an early stage and contribute to the transition from acute to chronic low back pain.     

Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis

Patients with sepsis in the ICU (intensive care unit) are characterized by skeletal muscle wasting. This leads to muscle dysfunction that also influences the respiratory capacity, resulting in prolonged mechanical ventilation. Catabolic conditions are associated with a general activation of the ubiquitin-proteasome pathway in skeletal muscle. The aim of the present study was to measure the proteasome proteolytic activity in both respiratory and leg muscles from ICU patients with sepsis and, in addition, to assess the variation of proteasome activity between individuals and between duplicate leg muscle biopsy specimens. When compared with a control group (n=10), patients with sepsis (n=10) had a 30% (P<0.05) and 45% (P<0.05) higher proteasome activity in the respiratory and leg muscles respectively. In a second experiment, ICU patients with sepsis (n=17) had a 55% (P<0.01) higher proteasome activity in the leg muscle compared with a control group (n=10). The inter-individual scatter of proteasome activity was larger between the patients with sepsis than the controls. We also observed a substantial intra-individual difference in activity between duplicate biopsies in several of the subjects. In conclusion, the proteolytic activity of the proteasome was higher in skeletal muscle from patients with sepsis and multiple organ failure compared with healthy controls. It was shown for the first time that respiratory and leg muscles were affected similarly. Furthermore, the variation in proteasome activity between individuals was more pronounced in the ICU patients for both muscle types, whereas the intra-individual variation between biopsies was similar for ICU patients and controls.