Serum markers of apoptosis decrease with age and cancer stage

Abstract

The physical manifestations of aging reflect a loss of homeostasis that effects molecular, cellular and organ system functional capacity. As a sentinel homeostatic pathway, changes in apoptosis can have pathophysiological consequences in both aging and disease. To assess baseline global apoptosis balance, sera from 204 clinically normal subjects had levels of sFas (inhibitor of apoptosis), sFasL (stimulator of apoptosis), and total cytochrome c (released from cells during apoptosis) measured. Serum levels of sFas were significantly higher while sFasL and cytochrome c levels were lower in men compared to women. With increasing age there was a decrease in apoptotic markers (cytochrome c) and pro-apoptotic factors (sFasL) and an increase in anti-apoptotic factors (sFas) in circulation. The observed gender differences are consistent with the known differences between genders in mortality and morbidity. In a separate cohort, subjects with either breast (n = 66) or prostate cancer (n = 38) exhibited significantly elevated sFas with reduced sFasL and total cytochrome c regardless of age. These markers correlated with disease severity consistent with tumor subversion of apoptosis. The shift toward less global apoptosis with increasing age in normal subjects is consistent with increased incidence of diseases whose pathophysiology involves apoptosis dysregulation.

Keywords: aging; apoptosis; cancer; cytochrome c; immunosenescence; serum markers.

Figure 1.. Serum sFas levels.

The levels of sFas in 204 normal subjects was determined by sandwich ELSA. The frequency distribution of the values across the subjects was analyzed (a). The levels of sFas by gender were plotted (b). The sample population was segregated by gender and the levels of serum sFasL as a function of donor age for female (c) and male (d) subjects were plotted.

Figure 2.. Serum sFasL levels.

The levels of sFasL in 204 normal subjects was determined by sandwich ELSA. The frequency distribution of the values across the subjects was analyzed (a). The levels of sFasL in all subjects as a function of gender were plotted (b). The sample population was segregated by gender and the levels of serum sFasL as a function of donor age for female (c) and male (d) subjects were plotted.

Figure 3.. Total cytochrome c assay.

A quantitative western blot assay was developed to measure total cytochrome c in serum. The assay employed denaturing and reducing conditions to disrupt cytochrome c binding to carrier proteins in serum. The assay utilized serial dilutions of purified cytochrome-c resolved by SDS PAGE and western blotting (a) to generate standard curves (b) by digitally imaging and quantifying the chemiluminescent signal and serum from men (c) and women (d) were analyzed in parallel. Standards and serum samples were analyzed in duplicate.

Figure 4.. Serum total cytochrome c levels.

The levels of total cytochrome c in 204 normal subjects were determined as depicted in Figure 3. The frequency distribution of the values across the subjects was analyzed (a). The levels of total cytochrome c in all subjects by gender was plotted (b). The sample population was segregated by gender and the levels of serum cytochrome c as a function of donor age for female (c) and male (d) subjects were plotted.

Figure 5.. Age and gender differences in serum sFas,.

sFasL and total cytochrome c levels. The serum levels of the apoptotic biomarkers were segregated by gender and by decade. Tukey box and whiskers plots (female clear boxes, male shaded boxes) of sFas (a), sFasL (b) and total cytochrome c (c) depicting the top, bottom, and line through the middle of the box correspond to the 75th percentile (top quartile), 25th percentile (bottom quartile), and 50th percentile (median) respectively. The error bar-like whiskers depict 1.5 x the interquartile range and the solid circles represent outliers. Comparisons between genders were performed conservatively by Mann Whitney U-test.

Figure 6.. Serum markers of apoptosis and tumor stage.

Subjects with breast cancer (a, c, e), or prostate cancer (b, d, f) were stratified by stage and the distribution of sFas (a, b), sFasL (c, d) and cytochrome c (e, f) stratified by staging was determined. The solid horzontal bars depict the median values. For breast cancer, stage I tumor size (T) < 2 cm across and cancer cells have not spread to axillary lymph nodes (N). For stage II, T < 2 cm across and the cancer has spread to the lymph nodes under the arm (N positive) or T is 2 to 5 cm and N is negative. In stage III, T > 5 cm or it has spread to other lymph nodes or tissues near the breast. Stage IV is metastatic cancer. The convention for prostate cancer staging was that in stage I, cancer is found in the prostate only. In stage II, cancer is more advanced than in stage I, but has not spread outside the prostate. In stage III, cancer has spread beyond the outer layer of the prostate to nearby tissues. Stage IV is characterized by distant metastasis. Comparison between group median values was performed by Mann Whitney t-test, where * = p < 0.05, ** = p < 0.005, *** = p < 0.0001. Numbers in parenthesis indicate number of subjects in each group.