Increased TNF-α and nitric oxide synthase-2 (NOS2) expression and activation of TNFR1 in skeletal muscle of cachectic patient. P values represent the significance of the mean group difference, as estimated by the mixed-effects model (see Supplemental Table S1). All 4 (C1–C4; from Table 1) control patients were analyzed (shown as open circles with a line indicating the mean), and 6 cachectic patients (A2, Ca1–Ca3, Ca6, and Ca8; from Table 1) were analyzed (shown as solid circles with a line indicating the mean). The entire group of cachectic patients could not be analyzed by this methodology because of insufficient tissue. Age, sex, and muscle biopsy location did not have statistical effects. All samples were run in triplicate, and replicate values were within an acceptable range of each other. Samples were 6 rectus abdominis and 4 vastus lateralis (Table 1). A: TNF-α mRNA was determined by RT-PCR as described in materials and methods. TNF-α mRNA was increased in skeletal muscle from cachectic patients (N = 6; samples described above) compared with control samples (N = 4; samples described above) (P < 0.05). B: TNF-α was determined by immunoblot in immunopurified TNFR1 as described in materials and methods. TNF-α was increased in skeletal muscle from cachectic patients (P < 0.001). Samples were those described above. Four representative control and 6 cachectic values are shown. IgG was used as a negative control. C: TNFR1 was determined by immunoblot in immunopurified TNFR1 as described in materials and methods. TNFR1 was unchanged in skeletal muscle from cachectic patients (P = 0.095). Samples were those described above. One representative control and 6 cachectic values are shown. IgG was used as a negative control. D: activated phosphorylated TNFR1 (pTNFR1) was determined by immunoblot in immunopurified TNFR1 as described in materials and methods. Phospho-TNFR1 was increased in skeletal muscle from cachectic patients (P < 0.001). Samples were those described above. Four representative control and 6 cachectic values are shown. IgG was used as a negative control. E: β-actin was determined by immunoblot in skeletal muscle lysates as described in materials and methods. β-Actin was unchanged in skeletal muscle from cachectic patients (not significant). Samples were those described above. Four representative control and 6 cachectic values are shown. F: NOS2 mRNA was determined by RT-PCR as described in materials and methods. NOS2 mRNA was increased in skeletal muscle from cachectic patients (N = 5; A2, Ca1–Ca3, and Ca6, Table 1) compared with control samples (N = 4, as described above) (P < 0.01). Samples were 6 rectus abdominis and 3 vastus lateralis (Table 1). Immunoblots quanified by optical density (OD).

Increased oxidative stress and NOS2 and decreased myosin and muscle creatine kinase (CKM) by immunohistochemistry in the skeletal muscle of cachectic patients. Summary of the analysis of 2 subjects with 3 replicates each. All of the measures were completely differentiated between subjects. That is, all replicates from one subject were less than the replicates from the other subject (or vice versa). The sample size was too small to support rigorous statistical analysis. We were not able to analyze the entire group of cachectic samples by this assay because of sample quantity limitations. Top: representative examples of skeletal muscle (vastus lateralis) immunohistochemistry of a control individual (C2, Table 1) and a patient afflicted by cancer cachexia (Ca9). We performed immunohistochemistry for malondialdehyde-protein adducts (MDA), NOS2, myosin, and CKM as described in materials and methods. There was an increase in the expression of MDA and NOS2 and a decrease in the expression of myosin and CKM in skeletal muscle of the cachectic patient. Negligible staining was observed in all immunohistochemical staining when the first antibody was omitted. Size bar represents 100 μm. Bottom: these findings by immunohistochemistry were quantified by using the Metamorph Offline program, Universal Imaging, product version 6.1. The quantitative measurements of 5 random fields each (×200) showed significant increases in MDA and NOS2 and decreases in myosin and CKM in the muscle of the cachectic patient. The sample size was too small to support rigorous statistical analysis.

Increased NOS2 and decreased myosin, CKM, myogenin and Jun-D by immunoblot in the skeletal muscle of cachectic patients. P values represent the significance of the mean group difference, as estimated by the mixed-effects model. All 4 (C1–C4; from Table 1) control patients were analyzed, and 6 cachectic patients (A1, Ca2, Ca3, Ca5, Ca7, and Ca8; from Table 1) were analyzed (horizontal lines indicate the means for each group). The entire group of cachectic patients could not be analyzed by this methodology because insufficient tissue. Age, sex, and muscle biopsy location did not have statistical effects. All samples were run in triplicate, and replicate values were within an acceptable range of each other. Samples were 5 rectus abdominis and 5 vastus lateralis (Table 1). Immunopurification and immunoblot were performed for NOS2 (A), myosin (B), CKM (C), myogenin (D), Jun-D (E), and β-actin (F) (control for immunopurification) by using specific antibodies in control and cachectic samples as described above. NOS2 was increased, whereas myosin, CKM, myogenin, and Jun-D were decreased in cachectic skeletal muscle samples compared with control samples. Four representative control and 6 cachectic values are shown. IgG was used as a negative control. Additional quantitation of these muscle proteins was obtained from the immunoblot, using the Alpha Ease FC program, Alpha Innotech Version 3.2.2. The expression of NOS2 (P < 0.001) was increased whereas the expressions of myosin (P < 0.001), CKM (P < 0.001), myogenin (P < 0.001), and Jun-D (P < 0.001) were decreased. These are representative data from 4 independent experiments.

Decreased Jun-D, myogenin, and CKM mRNA in skeletal muscle from cachectic patients. P values represent the significance of the mean group difference, as estimated by the mixed-effects model. All 4 (C1–C4; from Table 1) control patients were analyzed, and 6 cachectic patients (A1, Ca3–Ca5, Ca6, and Ca9, from Table 1) were analyzed (horizontal lines indicate the means for each group). The entire group of cachectic patients could not be analyzed by this methodology because of insufficient tissue. Age, sex, and muscle biopsy location did not have statistical effects. All samples were run in triplicate, and replicate values were within an acceptable range of each other. Samples were 6 rectus abdominis and 4 vastus lateralis (Table 1). RT-PCR amplification for Jun-D (A), myogenin (B), and CKM (C) was performed as described in materials and methods. Jun-D, myogenin, and CKM mRNA were decreased in skeletal muscle from cachectic patients [N = 6; as described above) compared with controls (N = 4; as described above)] (P < 0.001 for Jun-D; P < 0.005 myogenin; and P < 0.01 CKM). These are representative data from 5 independent experiments.

Jun-D is oxidatively modified, ubiquitinated, and decreased in skeletal muscle from cachectic patients. P values represent the significance of the mean group difference, as estimated by the mixed-effects model. The sample size was too small to support rigorous statistical analysis. We were not able to analyze the entire group of cachectic samples by this assay because of sample quantity limitations. Representative immunoblots obtained using specific antibodies against Jun-D (A) and myogenin (B) in immunopurified Jun-D from skeletal muscle lysates from control (Con) and cachexia (Pt.C) subjects representative of Con (N = 2; C2, C3, 1 rectus abdominis and 1 vastus lateralis, Table 1) and cachectic (N = 4; A1, Ca7, Ca9, and Ca10, 1 rectus abdominis and 3 vastus lateralis, Table 1) skeletal muscle samples. IgG was used a negative Con. Additional quantitation of these muscle proteins was obtained from the immunoblot, by using the Alpha Ease FC program, Alpha Innotech Version 3.2.2. The expression of Jun-D and myogenin in Jun-D immunoprecipitates was decreased. These data are representative of 3 independent experiments. Representative immunoblots obtained by using specific antibodies against ubiquitin (C), MDA (D), myogenin (E), and β-actin (F) (Con for immunopurification) in immunopurified myogenin from skeletal muscle lysates from Con and Pt.C subjects representative of Con (N = 2; as described in A and B) and cachectic (N = 4; as described in A and B) skeletal muscle samples. The ubiquitin antibodies reacted against proteins running as myogenin (top band) and Jun-D (bottom band). The MDA band corresponded to the Jun-D band. IgG was used a negative Con. Additional quantitation of these muscle proteins was obtained from the immunoblot, by use of the Alpha Ease FC program, Alpha Innotech Version 3.2.2. The expression of ubiquitin and MDA was increased and the expression of myogenin was decreased in myogenin immunoprecipitates. These data are representative of 3 independent experiments.