Mitochondrial Respiration in Human Colorectal and Breast Cancer Clinical Material Is Regulated Differently

Abstract

We conducted quantitative cellular respiration analysis on samples taken from human breast cancer (HBC) and human colorectal cancer (HCC) patients. Respiratory capacity is not lost as a result of tumor formation and even though, functionally, complex I in HCC was found to be suppressed, it was not evident on the protein level. Additionally, metabolic control analysis was used to quantify the role of components of mitochondrial interactosome. The main rate-controlling steps in HBC are complex IV and adenine nucleotide transporter, but in HCC, complexes I and III. Our kinetic measurements confirmed previous studies that respiratory chain complexes I and III in HBC and HCC can be assembled into supercomplexes with a possible partial addition from the complex IV pool. Therefore, the kinetic method can be a useful addition in studying supercomplexes in cell lines or human samples. In addition, when results from culture cells were compared to those from clinical samples, clear differences were present, but we also detected two different types of mitochondria within clinical HBC samples, possibly linked to two-compartment metabolism. Taken together, our data show that mitochondrial respiration and regulation of mitochondrial membrane permeability have substantial differences between these two cancer types when compared to each other to their adjacent healthy tissue or to respective cell cultures.

Figure 1

Assessment of state 2 respiration rates of the permeabilized HCC, HBC, and normal adjacent tissue samples in the presence of different combinations of respiratory substrates (5 mM glutamate, 2 mM malate, and 10 mM succinate). Bars are SEM, n = 8 for colon samples, and n = 12 for breast tissue samples, ^∗p < 0.05.

Figure 2

(a) Oxygraphic analysis of the functioning of complex I in skinned tissues from patients with HBC or HCC; here, V_Glut/V_Succ is the ratio of ADP-stimulated respiration rate in the presence of 5 mM glutamate and 2 mM malate (activity of complex I) to ADP-stimulated respiration rate in the presence of 50 μM rotenone and 10 mM succinate (activity of complex II). (b) V_Succ/V_COX is the ratio of complex II respiration rate to complex IV respiration rate. Data shown as mean ± SEM; n = 7 for colon [28] and breast tissue samples [26], ^∗p < 0.05.

Figure 3

Quantitative analysis of the expression levels of the respiratory chain complexes in HCC and normal tissue samples (a) along with a representative Western blot image (b). Protein levels were normalized to total protein staining by Coomassie blue; data shown as mean ± SEM of 5 independent experiments.

Figure 4

FCCs for ATP synthasome and RC complexes as determined by MCA. Two ways of electron transfer were examined: NADH-dependent and succinate-dependent electron transfers, and respective sums of FCCs are calculated as the last bars. Data for HBC is published before in [26], except for complex II with atpenin A5. Isolated mucosal tissue was used for colon control.

Figure 5

(a) Respiration rates for clinical samples of luminal-A and triple negative HBC subtypes in the presence of 5 mM glutamate or 5 mM pyruvate; n = 13/12 for luminal-A and n = 7/8 for triple negative subtypes, respectively. (b) Respiratory rates for luminal-A type MCF-7 and triple negative MDA-MB-231 cells in the presence of 5 mM glutamate or 5 mM pyruvate; n = 3 for each measurement; ^∗p < 0.05, ^∗∗p < 0.005.

Figure 6

(a) Dependence of maximal rate of mitochondrial respiration (V_max) compared with the HCC at different stages. Stage I was calculated as the mean of 13 patients, IIA, IIB - 13 patients, IIIB-4 patients, IIIC-3 patients and IVB-1 patient. Control colon tissue is obtained from 34 patients. Maximal respiration rate V_max is compared with that in control tissue. Bars are SEM; ^∗∗p < 0.005. (b) V_max in HCC patients based on disease state in follow-up setting. Seven patients out of 32 are confirmed to have succumbed to HCC (V_max = 3.19 ± 0.34); 25 patients out of 32 stay in remission (V_max = 1.70 ± 0.17), ^∗∗∗p < 0.001.

Figure 7

(a) Dependences of normalized respiration rate values for HCC (dotted line), HBC (solid line), and healthy colon tissue samples (dashed line); double reciprocal Lineweaver–Burk plots. Samples from 32 patients with breast cancer and 10 patients with colorectal cancer were examined. (b) ADP-dependent respiration in healthy colon mucosa and smooth muscle tissue samples (Michaelis–Menten curve, n = 8). Here, Vo and V_max are rates of basal and maximal ADP-activated respiration, respectively.

Figure 8

Mitochondrial alterations in HCC and HBC tissue cells. Mitochondrial interactosome is a large supercomplex consisting of ATP synthasome, VDAC, mitochondrial kinases like adenylate kinase, hexokinase or mitochondrial creatine kinase (MtCK), and respiratory chain (super)complexes. Here, the octameric MtCK characteristic is shown for the striated muscles and also as the possible component of the MI in the healthy colon [–110]. The complex of VDAC together with other proteins controls the exchange of adenine nucleotides and regulates energy fluxes between mitochondrial and cytosolic compartments. Changes in the structure and function of MI are the important parts of cancer mitochondrial metabolism.