Effect of the specific proteasome inhibitor bortezomib on cancer-related muscle wasting

Abstract

Background: Muscle wasting, a prominent feature of cancer cachexia, is mainly caused by sustained protein hypercatabolism. The enhanced muscle protein degradation rates rely on the activity of different proteolytic systems, although the Adenosine triphosphate (ATP)-ubiquitin-proteasome-dependent pathway and autophagy have been shown to play a pivotal role. Bortezomib is a potent reversible and selective proteasome and NF-κB inhibitor approved for the clinical use, which has been shown to be effective in preventing muscle wasting in different catabolic conditions. The aim of the present study has been to investigate whether pharmacological inhibition of proteasome by bortezomib may prevent skeletal muscle wasting in experimental cancer cachexia.

Methods: Cancer cachexia was induced in rats by intraperitoneal injection of Yoshida AH-130 ascites hepatoma cells and in mice by subcutaneous inoculation of C26 carcinoma cells. Animals were then further randomized to receive bortezomib. The AH-130 hosts were weighted and sacrificed under anaesthesia, on Days 3, 4, 5, and 7 after tumour inoculation, while C26-bearing mice were weighted and sacrificed under anaesthesia 12 days after tumour transplantation. NF-κB and proteasome activation, MuRF1 and atrogin-1 mRNA expression and beclin-1 protein levels were evaluated in the gastrocnemius of controls and AH-130 hosts.

Results: Bortezomib administration in the AH-130 hosts, although able to reduce proteasome and NF-κB DNA-binding activity in the skeletal muscle on Day 7 after tumour transplantation, did not prevent body weight loss and muscle wasting. In addition, bortezomib exerted a transient toxicity, as evidenced by the reduced food intake and by the increase in NF-κB DNA-binding activity in the AH-130 hosts 3 days after tumour transplantation. Beclin-1 protein levels were increased by bortezomib treatment in Day 3 controls but were unchanged on both Days 3 and 7 in the AH-130 hosts, suggesting that an early compensatory induction of autophagy may exist in healthy but not in tumour-bearing animals. Regarding C26-bearing mice, bortezomib did not prevent as well body and muscle weight loss 12 days after tumour implantation.

Conclusions: The results obtained suggest that proteasome inhibition by bortezomib is not able to prevent muscle wasting in experimental cancer cachexia. Further studies are needed to address the issue whether a different dosage of bortezomib alone or in combination with other drugs modulating different molecular pathways may effectively prevent muscle wasting during cancer cachexia.

Keywords: Bortezomib; Cancer cachexia; Muscle wasting; Proteasome.

Figure 1

Bortezomib does not prevent body and gastrocnemius muscle weight loss in AH‐130‐bearing rats. (A) Body and (B) gastrocnemius weight were evaluated in controls (C) and tumour‐bearing rats (AH‐130) treated or untreated with bortezomib (BTZ) at Day 3 (d3) and Day 7 (d7) after AH‐130 cell inoculation. Results (means ± SD) are expressed as percentage of controls (C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Figure 2

Effects of bortezomib on liver and spleen weight. (A) Liver and (B) spleen weight were evaluated in controls (C) and tumour‐bearing rats (AH‐130) treated or untreated with bortezomib (BTZ) at Day 3 (d3), Day 4 (d4), Day 5 (d5), and Day 7 (d7) after AH‐130 cell inoculation. Results (means ± SD) are expressed as percentage of controls (C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8). *P < 0.05, **P < 0.01 vs. control; ^¶ P < 0.05 vs. C + BTZ.

Figure 3

Effects of bortezomib on food and water intake. (A) Food intake and (B) water intake were evaluated daily in control (C) and tumour‐bearing rats (AH‐130) treated or untreated with bortezomib (BTZ). C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8.

Figure 4

Effects of bortezomib on AH‐130 cell number. Total cell number were evaluated in tumour‐bearing rats (AH‐130) treated or untreated with bortezomib (BTZ) at Day 3 (d3), Day 4 (d4), Day 5 (d5), and Day 7 (d7) after AH‐130 cell inoculation. Results are expressed as means ± SD (C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8). *P < 0.05, ***P < 0.001 vs. AH‐130.

Figure 5

Bortezomib modulates NF‐κB nuclear translocation in the gastrocnemius of the AH‐130 hosts. (A) Densitometric analysis and (B) representative pattern of NF‐κB DNA‐binding activity measured by electrophoretic mobility shift assay in the gastrocnemius of control (C) and tumour‐bearing rats (AH‐130) treated or untread with bortezomib (BTZ) at Day 3 (d3) and Day 7(d7) after AH‐130 cell inoculation. Results (means ± SD) are expressed as percentage of controls (C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8). *P < 0.05 vs. control, §P < 0.05, §§P < 0.01 vs. AH‐130.

Figure 6

Bortezomib does not modulate atrogin‐1 and MuRF1 mRNA levels in the gastrocnemius of the AH‐130 hosts. mRNA levels for (A) atrogin‐1 and (B) MuRF1 were determined by semi‐quantitative RT‐PCR in the gastrocnemius of controls (C) and tumour‐bearing rats (AH‐130) treated or untread with bortezomib (BTZ) at Day 3 (d3) and Day 7 (d7) after AH‐130 cell inoculation. Results (means ± SD) are expressed as percentage of controls (C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8). **P < 0.01, ***P < 0.001 vs. control.

Figure 7

Bortezomib does not modulate beclin‐1 protein levels in the gastrocnemius of the AH‐130 hosts. Beclin‐1 protein levels in gastrocnemius muscle of controls (C) and tumour‐bearing rats (AH‐130) treated and untreated with bortezomib (BTZ) at Day 3 (d3) and Day 7 (d7) after AH‐130 cell inoculation were evaluated by western blotting. α‐tubulin was used as loading control. Densitometric quantification are shown in the figure, and results (means ± SD) are expressed as percentage of controls (C and C + BTZ: n = 4; AH‐130, AH‐130 + BTZ: n = 8). *P<0.05 vs. control.

Figure 8

Bortezomib does not affect food intake and does not prevent body and gastrocnemius muscle weight loss in C26‐bearing mice. (A) Daily food intake, (B) body weight, and (C) gastrocnemius weight were evaluated in control (C) and tumour‐bearing mice (C26) treated or untreated with bortezomib (BTZ) 12 days after C26 cell inoculation. Results (means ± SD) are expressed as percentage of controls (controls: n = 7; C26: n = 8; C26 + BTZ: n = 7). **P < 0.001 vs. control.