Relationships between expression of BCS1L, mitochondrial bioenergetics, and fatigue among patients with prostate cancer

Figures & data

Table 1 Demographic and clinical characteristics of sample (N=52)

Variables	Participants		P-value
	XRT subjects (N=35)	Controls (N=17)
Mean (± SEM) or N (%)	Mean (± SEM) or N (%)
Mean age, years	67.7 (±1.4)	63.7 (±1.7)	0.09
Ethnicity:
Caucasian	21 (60%)	15 (88%)
African-American	14 (40%)	1 (6%)
Asian		1 (6%)
Marital status
Married	22 (63%)	15 (88%)
Widowed	6 (17%)
Single	3 (9%)
Divorced/separated	4 (11%)	2 (12%)
Employment status
Employed	14 (40%)	13 (76%)
Retired	19 (54%)	4 (24%)
Disabled	2 (6%)
T-stage
T1c	31 (88%)	16 (94%)
T2a-c	2 (6%)	1 (6%)
T3a	2 (6%)
Gleason Score
7 Jun	21 (60%)	16 (94%)
10 Aug	14 (40%)	1 (6%)
Karnofsky performance scale	88.6 (±0.8)	90.0 (±0.8)	0.62
PSA (ng/mL)
Baseline	12.6 (±2.12)	4.7 (±0.6)	0.02
Endpoint	0.7 (±0.95)	4.2 (±0.6)	0.01
Hemoglobin (mg/dL)			0.46
Baseline	14.4 (±0.3)	15.3 (±0.7)
Endpoint	13.9 (±0.4)
Hematocrit (%)			0.22
Baseline	43.4 (±0.8)	45.6 (±1.7)
Endpoint	41.1 (±0.9)
Depression (HAM-D)
Baseline	1.3 (±0.3)	0.5 (±0.3)	0.12
Endpoint	1.2 (±0.3)	0.2 (±0.1)	0.09
Total EBRT dosage (Gy)
79.2–81	24 (68%)	None
100	11 (32%)

Notes: Controls are men with nonmetastatic prostate cancer and not receiving any treatment are placed in active surveillance.

Abbreviations: SEM, standard error of the mean; PSA, prostate- specific antigen; HAM-D, Hamilton Depression Rating Scale; XRT, external beam radiation therapy.

Figure 1 Changes in fatigue score between patients undergoing radiation therapy (N=35) and active surveillance (N=17). Fatigue was measured by the revised Piper Fatigue Scale (rPFS).

Notes: X-axis indicates time points: day 0=prior XRT; day 21=day 21 of XRT, midpoint; day 42=completion of XRT, endpoint. Y-axis represents revised Piper Fatigue score. Fatigue score was significantly increased at day 21 (t=2.06, P=0.03) and day 42 (t=2.71, P=0.002) in patients with XRT; additionally, there was a significant difference in fatigue scores between XRT and AS groups over time. ^aRefers to a significant difference compared to day 0 time point of the same group. ^bRefers to a significant difference compared to the AS group at the same time point. *P<0.05; **P<0.01. ^a*^b*Represents a significant change in fatigue score (P<0.05) in patients receiving XRT and a significant difference in fatigue score (P<0.05) between XRT and AS groups. ^a**^b*Represents a significant change in fatigue score (P<0.01) in patients receiving XRT, and a significant difference in fatigue score (P<0.05) between XRT and AS groups.

Abbreviations: XRT, external beam radiation therapy; AS, active surveillance.

Table 2 Integrated mitochondrial function in peripheral blood mononuclear cells of patients with prostate cancer

Variables	Prostate cancer patients with radiation therapy or on active surveillance
	Baseline/day 0		Midpoint/day 21		Endpoint/day 42
	XRT (N=33)	AS (N=17)	XRT (N=34)	AS (N=17)	XRT (N=35)	AS (N=17)
Cell number (10^6/mL)	1.8±0.19	2.0±0.26	1.5±0.33	2.1±0.26	1.6±0.48	1.9±0.39
Protein concentration (mg/mL)	0.51±0.25	0.49±0.32	0.50±0.18	0.50±0.28	0.55±0.22	0.51±0.39
Protocol 1	(pmol O2/sec/10^6 cells) Mean ± SEM
Intact cell	3.55±0.23	3.54±0.29	3.61±0.21	4.06±0.32	3.31±0.18	3.75±0.34
Intact cell (malate+pyruvate)	4.49±0.26	4.38±0.32	4.51±0.29	4.84±0.36	4.41±0.26	4.63±0.31
Leak (digitonin)	1.24±0.20	1.02±0.23	1.26±0.25	1.70±0.46	1.18±0.19	0.83±0.21
OXPHOS (complex I-linked)	4.67±0.35	4.59±0.77	4.51±0.44	5.12±0.39	5.19±0.45	6.04±0.58
OXPHOS (complex I+II-linked)	14.21±0.76	13.16±1.21	^a*11.95±0.7	13.57±1.15	^a*b*12.29±0.9	15.15±1.35
Uncoupled CI+II-linked, maximal oxidative capacity (uncoupler)	15.45±0.89	14.23±1.13	^a*13.25±1.0	14.25±1.24	^b*13.85±0.9	16.11±1.44
Uncoupled complex I	4.96±0.46	5.11±0.59	5.01±0.77	4.82±0.71	4.91±0.47	6.03±0.59
Uncoupled complex II	8.74±0.64	8.06±0.93	^a*6.17±0.53	8.18±0.84	7.15±0.66	8.86±1.57
Uncoupled complex IV	229.5±15.9	247.9±20.8	245.7±13.5	251.1±30.9	220.6±10.8	272.9±17.6
Citrate synthase	3.58±0.34	2.98±0.59	3.31±0.29	3.27±0.64	4.09±0.63	2.95±0.47
Acceptor control ratio	4.53±0.58	5.05±0.72	4.12±0.64	5.36±1.50	4.68±0.51	5.41±1.02
Uncoupled control ratio	1.09±0.25	1.10±0.43	1.15±0.62	1.06±0.03	1.07±0.25	1.03±0.34
Protocol 2
Intact cell	3.36±0.21	3.72±0.27	3.52±0.32	3.53±0.31	3.19±0.21	3.65±0.29
Intact cell (malate+palmitoylcarnitine)	3.95±0.25	4.17±0.23	4.29±0.41	4.13±0.25	3.97±0.21	4.43±0.33
Leak (digitonin)	1.20±0.21	1.15±0.22	1.70±0.42	1.37±0.41	1.33±0.18	1.23±0.32
OXPHOS (fatty acid oxidation)	3.76±0.29	3.31±0.27	3.84±0.33	3.35±0.37	3.64±0.23	3.46±0.23
OXPHOS (coupled complex III)	21.43±1.03	19.46±0.84	^a*19.05±0.84	19.31±1.91	^a*b*19.01±1.18	21.26±1.75
Uncoupled complex III (uncoupler)	20.26±1.12	18.36±1.39	^a*17.74±1.04	18.46±1.52	^a*18.44±1.38	19.95±1.52
Citrate synthase	4.91±0.55	2.60±0.41	4.95±0.58	2.97±0.47	3.97±0.54	2.79±0.39
Acceptor control ratio	4.24±0.68	4.36±0.50	^b*3.65±0.63	6.31±1.75	3.94±0.49	5.21±0.79
Uncoupled control ratio	0.96±0.22	0.95±0.25	0.93±0.31	0.90±0.34	0.98±0.02	0.94±0.32

Notes: Data are subtracted from non-mitochondrial respiration (antimycin A respiration for protocol 1, rotenone and antimycin A respiration for protocol 2). Data are presented as mean ± standard error. Integrated mitochondrial function was measured as oxidative phosphorylation. ^aSignificant difference compared to day 0 time point of the same group. ^bSignificant difference compared to the AS group at same time point. *P<0.05.

Abbreviations: XRT, external radiation therapy; AS, active surveillance; SEM, standard error of the mean; OXPHOS, oxidative phosphorylation; CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV.

Figure 2 Changes in mitochondrial oxidative phosphorylation (OXPHOS, complex I+II linked, uncoupled complex I+II-linked, coupled complex III, and uncoupled complex III) over time between patients with radiation therapy (N=33) and active surveillance (N=15). ₂⁶pp

Notes: X-axis indicates time points and Y-axis represents cell respiration rate (pmol O₂/sec/106 cells). A: OXPHOS-complex I+II linked oxidation was measured as ADP-stimulated respiration with malate, pyruvate, glutamate, and succinate as substrates, reflecting the convergence of complex I and II. B: Uncoupled complex I+II was obtained with addition of the uncoupler, FCCP representing maximal oxidative capacity. C: Coupled complex III rate was measured as the ADP-stimulated respiration with DHQ, a substrate for complex III. D: Uncoupled complex III was determined after the addition of the uncoupler, FCCP. ^aRefers to a significant difference compared to day 0 time point of the same group. ^bRefers to a significant difference compared to the AS group at the same time point. *P<0.05; **P<0.01. ^a*b*Represents a significant change in fatigue score (P<0.05) in patients receiving XRT and a significant difference in fatigue score (P<0.05) between XRT and AS groups.

Abbreviations: OXPHOS, oxidative phosphorylation; XRT, external beam radiation therapy; AS, active surveillance; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; DHQ, duroquinol.

Table 3 Electron-transport chain enzymatic activity in peripheral blood mononuclear cells

Variables	Prostate cancer patients with radiation therapy or on active surveillance
	Baseline/day 0		Midpoint/day 21		Endpoint/day 42
	XRT (N=35)	AS (N=17)	XRT (N=34)	AS (N=15)	XRT (N=35)	AS (N=17)
Protein concentrations (mg/mL)	4.29±0.41	3.87±0.29	4.33±0.34	3.94±0.25	4.24±0.29	4.59±0.35
nmol/min/mg protein	Mean ± SEM
Rotenone-sensitive CI/III	13.7±0.89	12.93±1.27	12.35±0.88	13.75±1.42	12.85±0. 98	12.39±0.74
Antimycin A-sensitive CII/III	10.3±0.63	11.18±0.92	9.69±0.55	11.24±0.82	^a*b*8.92±0.59	11.01±0.85
Succinate dehydrogenase (SDH)	4.19±0.32	3.69±0.45	3.89±0.25	4.66±0.46	3.38±0.35	3.96±0.36
TTFA-sensitive CII	2.01±0.14	1.41±0.19	1.79±0.13	2.04±0.19	1.41±0.14	1.88±0.19
TTFA-sensitive CII+Q1	9.28±0.58	8.39±0.89	^a*8.37±0.43	10.18±0.62	^a*7.65±0.51	9.09±0.56
Antimycin A-sensitive CIII	42.44±3.07	43.56±2.83	^a*b*39.8±1.62	45.63±2.78	^a*b*40.66±1.79	45.71±2.85
Cytochrom C Oxidase CIV (k^−1/mg protein)	1.56±0.98	1.59±0.16	1.81±0.13	1.91±0.15	1.51±0.96	1.76±0.15
Mitochondrial marker Citrate synthase	52.63±2.69	50.89±4.68	50.98±2.48	52.08±4.34	^a*b*48.95±2.75	52.43±5.48
Cytoplasmic marker lactate dehydrogenase (LDH)	502.1±26.1	568.1±43.7	514.1±29.2	591.7±30.1	557.7±29.1	580.8±42.5

Notes: Data are presented as mean ± standard error. ^aSignificant difference compared to day 0 time point of the same group; ^bSignificant difference compared to the AS group at same time point. *P<0.05.

Abbreviations: SEM, standard error of the mean; Rotenone-sensitive CI/III, rotenone-sensitive linked activity of complex I and III; antimycin A-sensitive CII/III, antimycin A-sensitive linked activity of complex II and III; SDH, succinate dehydrogenase; TTFA-sensitive CII, complex II activity as the thenoyltrifluoroacetone (TTFA)-sensitive without CoQ₁ or with CoQ₁; antimycin A-sensitive CIII, complex III activity; cytochrome c oxidase; CIV, cytochrome c oxidase, complex IV activity; LDH, lactate dehydrogenase.

Figure 3 Changes in electron-transport chain complex enzymatic activity in mononuclear cells in patients with radiation therapy (N=33).

Notes: CII was measured as TTFA-sensitive DCPIP reductase; total complex II determined with coenzyme Q₁ added (CII+Q₁); CIII, measured as antimycin A-sensitive decylubiquinol cytochrome c reductase; CS is mitochondrial marker; SDH was used to measure the first 2 subunits of complex II and served as a membrane-bonded mitochondrial marker enzyme; LDH as a cytoplasm marker. *P<0.05.

Abbreviations: NADH, nicotinamide adenine dinucleotide; NCR, rotenone-sensitive NADH cytochrome c reductase; SCR, antimycin A-sensitive succinate cytochrome c reductase; CII, complex II activity; TTFA, thenoyltrifluoroacetone; DCPIP, dichlorophenol-indophenol; CII+Q1, complex II with coenzyme Q₁; CIII, complex III activity, measured as antimycin A-sensitive decylubiquinol cytochrome c reductase; CIV, complex IV (cytochrome c oxidase) activity; CS, citrate synthase, mitochondrial marker; SDH, succinate dehydrogenase; LDH, lactate dehydrogenase.

Figure 4 Changes in ETC complex III between patients with radiation therapy (N=33) and active surveillance (N=15).

Notes:Y-axis represents cell enzyme activity (nmol/min/mg protein). ETC CIII enzymatic activity, which was measured as antimycin A-sensitive decylubiquinol cytochrome c reductase. ETC CIII activity was significantly decreased at day 21 (t=−2.64, P=0.044) and day 42 (t=−1.78, P=0.047) in patients with XRT; additionally, there was a significant difference in CIII activity between XRT and AS groups over time. ^aRefers to a significant difference compared to day 0 time point of the same group. ^bRefers to a significant difference compared to the AS group at the same time point. *P<0.05.

Abbreviations: ETC, electron transport chain; CIII, complex III activity; XRT, external beam radiation; AS, active surveillance.

Figure 5 Differences in gene expression of BCS1L at day 21 and day 42 between patients undergoing radiation therapy (XRT, N=33) and active surveillance (AS, N=13).

Notes: Left figure indicates the expression of BCS1L gene. Y-axis represents expression values assayed by monitoring in real-time polymerase chain reaction calculated as fold change. The expression of BCS1L was significant different between patients undergoing XRT and AS (P<0.05). Furthermore, BCS1L was significantly downregulated at day 21 (fold change=−1.76, P=0.03) and day 42 (fold change=−1.87, P=0.02) in XRT patients compared to day 0. ^aRefers to a significant difference compared to day 0 time point of the same group. ^bRefers to a significant difference compared to the AS group at the same time point. *P<0.05.

Abbreviations: BCS1L, BC1 (ubiquinol-cytochrome c reductase) synthesis-like; XRT, external beam radiation therapy; AS, active surveillance.

Figure 6 ETC complex III activity without and with normalization of citrate synthase (A and B) and CII+Q₁ (C) and SCR (D) with normalization of citrate synthase between patients undergoing XRT (N=35) and AS (N=15). (A) CIII without normalization of CS; (B) CIII with normalization of CS; (C) CII+Q₁ with normalization of CS; (D) SCR with normalization of CS.

Notes:^aSignificant difference compared to day 0 time point of the same group. ^bSignificant difference compared to the AS group at the same time point. *P<0.05. CIII, measured as antimycin A-sensitive decylubiquinol cytochrome c reductase; CII, measured as the TTFA-sensitive succinate-2,6 DCPIP reductase and total CII determined with coenzyme Q added (CII+Q₁); SCR, antimycin A-sensitive succinate cytochrome c reductase was used to measure the linked activity of CII and CIII.

Abbreviations: CIII, complex III activity; CS, citrate synthase; CII, complex II activity; TTFA, thenoyltrifluoroacetone; DCPIP, dichlorophenol-indophenol; CII+Q1, complex II with coenzyme Q₁; SCR, succinate cytochrome c reductase; XRT, patients undergoing radiation therapy; AS,  patients undergoing active surveillance.