Expression of NF-kappaB and IkappaB proteins in skeletal muscle of gastric cancer patients

Abstract

The mechanisms eliciting cancer cachexia are not well understood. Wasting of skeletal muscle is problematic because it is responsible for the clinical deterioration in cancer patients and for the ability to tolerate cancer treatment. Studies done on animals suggest that nuclear factor of kappa B (NF-kappaB) signalling is important in the progression of muscle wasting due to several types of tumours. However, there are no published studies in humans on the role of NF-kappaB in cancer cachexia. In this project, we studied the rectus abdominis muscle in patients with gastric tumours (n=14) and in age-matched control subjects (n=10) for markers of NF-kappaB activation. Nuclear levels of p65, p50 and Bcl-3 were the same in both groups of subjects. However, phospho-p65 was elevated by 25% in the muscles of cancer patients. In addition, expression of the inhibitor of kappa B alpha (IkappaBalpha) was decreased by 25% in cancer patients. Decreased expression of IkappaBalpha reflects its degradation by one of the IkappaBalpha kinases and is a marker of NF-kappaB activation. Interestingly, there was no correlation between the stage of cancer and the extent of IkappaBalpha decrease, nor was there a correlation between the degree of cachexia and decreased IkappaBalpha levels. This suggests that the activation of NF-kappaB is an early and sustained event in gastric cancer. The work implicates the NF-kappaB signalling in the initiation and progression of cancer cachexia in humans and demonstrates the need for additional study of this pathway; it also recommends NF-kappaB signalling as a therapeutic target for the amelioration of cachexia as has been suggested from studies done on rodents.

Fig. 1

Protein expression using immunobloting of nuclear fractions isolated from control and cancer patients. Average signal intensity values are plotted for (A) p65 protein expression, (B) p50 protein expression, and (C) Bcl-3 protein expression. 40ug protein loaded per lane. All signals were normalized to histone H2B (not shown). Values are mean ± SEM. n=6 control and n=10 cancer patients. No statistically significant differences were found between control and cancer subjects.

Fig. 2

Total muscle p65 and phospho-p65 expression isolated from control (n=5) and cancer patients (n=6). (A) whole muscle p65 expression and (B) phospho-p65 expression. Signals were normalized to α-tubulin. (C) Representative samples from immunoblots of phopho-p65, p65, and α-tubulin from samples in panels A and B. Hela lysates were used as a positive control. 70μg protein loaded per lane. * indicates significantly increased compared to control value (P<0.05).

Fig. 3

Cytosolic IκBα expression using immunoblotting in control and cancer patients. (A) Cancer patients showed a significant 25% decrease in IκBα expression compared to age-matched controls (P<0.01). 50μg protein loaded per lane. (B) Examples of signals for IκBα (top panel) and α-tubulin (lower panel), the latter used for signal normalization. HeLa lysates are positive control. (C) Individual values plotted for IκBα expression for control and cancer patients parsed by cancer stage. (D) Individual values for IκBα expression for control and cancer patients parsed by degree of cachexia. No differences in IκBα expression due to either cancer stage or amount of weight loss. n=10 controls, n=14 cancer patients.