Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line

Abstract

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.

Figure 1. Mitochondrial Respiratory Profile of ovarian cancer cell lines.

A. Oxygen Consumption Rate (OCR) measurements were obtained over time (min) using an extracellular flux analyzer (Seahorse Bioscience). The mitochondrial stress test was used to obtain bioenergetics parameters, by adding the ATP synthase inhibitor Oligomycin A (O, 1 µM), to derive ATP-linked OCR, FCCP (F, 750 nM) to uncouple the mitochondria for maximal OCR, and Antimycin A (A, 1 µM). B. Basal mitochondrial OCR of ovarian cancer cell lines was derived by subtracting non-mitochondrial OCR (remaining OCR after Antimycin A addition). C. ATP-linked OCR was derived as the difference between basal and Antimycin A inhibited OCR. D. OCR attributed to proton leak was calculated as the difference between OCR following Oligomycin A inhibition and OCR following Antimycin A inhibition. E. Maximum OCR was stimulated by FCCP addition. F. The respiratory reserve capacity was calculated as the difference between maximal and basal OCR. Graphs B-F represent data from one replicate experiment (n = 5, ANOVA, Tukey's post test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 statistically significant compared to NOSE007; #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 statistically significant compared to OVCA420). OCCC cell lines are highlighted in black and a normal epithelial cell line in white. G. Average Respiratory State_Apparent was calculated from 3–4 replicative experiments using: 4−(Basal OCR−Oligo OCR)/(FCCP OCR−Oligo OCR).

Figure 2. Extracellular Acidification Rate (ECAR) profile of Ovarian Cancer Cell lines.

A. Basal ECAR levels were measured using an extracellular flux analyzer (Seahorse Bioscience). B. Maximal ECAR was stimulated by addition of 1 µM Oligomycin A. C. Glycolytic Reserve Capacity was calculated as the difference between maximal and basal OCR. Data from one replicative experiment is shown (n = 5, ANOVA, Tukey's post test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 statistically significant compared to NOSE007; #p<0.05, ##p<0.01, ###p<0.001 statistically significant compared to OVCA420).

Figure 3. Bioenergetics Profile of Ovarian Cancer cell lines.

A. Plotting Basal ECAR and OCR levels provides a snap-shot of the bioenergetics profiles of the ovarian cancer cell lines studied. OCCC cell lines ES-2 and TOV-21-G as well as OVCAR3 cells display high glycolysis and Oxidative phosphorylation and are therefore classified as highly energetic cells. B. Plotting Glycolytic Reserve against Respiratory reserve indicates that some cells appear to be operating close to their maximal rate, such as ES-2 cells, whereas others have high respiratory reserve (NOSE007) or high glycolytic Reserve (DOV-13, OVCA429). OCCC cell lines are labeled as black triangles, other EOC cell lines as grey circles (OVCA420 as circle with X), and the normal epithelial cell lines NOSE007 as a white square. Data in A and C represents the average of mean OCR and ECAR readings from 3–4 experimental replicates C. Spheroid formation correlates with bioenergetic signature of ovarian cancer cells. Cells were seeded into ULA plates and size (area of spheroid aggregate in pixels) plotted (n = 6).

Figure 4. Chemoresistance Profile of Ovarian Cancer cells.

Cells were treated with indicated doses for 72A. Cisplatin, B. Paclitaxel, C. 2-Deoxyglucose (2-DG), D. Resveratrol, E. Metformin, F. Rotenone, and G. Combination treatment of low dose Rotenone (R; 0.1 µM) and 2-DG (2 mM). Data from one replicative experiment is shown (n = 5-6, A–F: ANOVA, Tukey's post test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 significantly less cell viability compared to NOSE007 at same treatment concentration; G: Student's T-test, statistically significant compared to each cell line treatment with rotenone alone [*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001], or 2-DG alone [#p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001]). Asterisks highlight OVCA420 cells, which have defective mitochondrial respiration. H. Expression of GLUT-1 and HNF-1β message in Ovarian cancer cells, as assessed by semi-quantitative real time RT-PCR. Expression was correlated to levels in NOSE007 cells (n = 3; ND  =  no measurable signal detected).

Figure 5. OVCA420 cells have defective mitochondria.

A. Mitotracker red staining reveals that OVCA420 cells display defects in mitochondrial morphology compared to NOSE007, ES-2 and OVCA433 cells. Representative images show fragmented mitochondria (a&b, arrows) and the presence of large globular aggregates (c&d, arrows) in OVCA420 cells. B. Assessment of mitochondrial to nuclear DNA ratios by comparison of mtDNA 16S Ct/nDNA 18S Ct ratios in total DNA from ovarian cancer cell lines, using real time PCR analysis. C. OVCA420 cells display enhanced expression of the fission protein Drp1 D. Densitometric quantification of the full length and Drp1 splice variant (n = 3, ANOVA, Tukey's post test *p<0.05, ****p<0.0001 significant difference compared to NOSE007; #p<0.05, ####p<0.0001 significant difference compared to OVCA420). E. OVCA420 cells display a decrease in mitochondrial membrane potential as evident by the lack of red JC-1 aggregate accumulation and higher staining for JC-1 green monomers. F. Quantification of red/green JC-1 staining indicative of membrane potential (n = 35 cells, ****p<0.0001, t-test).

Figure 6. OVCA420 cells with defective mitochondria lack ability to respond to hypoxia.

A. OVCA420 cells do not increase their clonogenic survival in response to hypoxia, unlike OVCA433 and ES-2 cells. B. Clonogenicity was assessed 10 days following incubation at either normoxic (21% O₂) or hypoxic (1% O₂) conditions and survival fraction quantified (n = 6, *p<0.05, **p<0.01, t-test). NOSE007 did not form colonies under either condition C. OVCA420 cells are unable to maintain cell viability under hypoxic conditions. Cell viability was assessed 72 hours following culturing under normoxic (21% O₂) or hypoxic (1% O₂) conditions and assessed by crystal violet uptake assay (n = 6, *p<0.05, t-test). D. OVCA420 cells display abrogated HIF-1α protein stabilization in response to hypoxia. Cells were exposed for 2 hours to 1% O₂ and HIF-1α levels visualized following immediate lysis and immunoblotting. Data were quantified by densitometry analysis relative to actin loading control and normalized to HIF-1α levels observed under normoxia (E).