Mitochondria are highly dynamic organelles that are involved in the regulation of cellular metabolism, redox regulation, and programmed cell death/apoptosis. Mitochondria constitute the principal energy source of the cell and convert nutrients into energy through cellular respiration [1
-stilbene) (Resv) is a naturally occurring polyphenol found in a high number of unrelated plant species including grapes, cranberries and peanuts, and also in a number of herbal remedies [2
]. Resv shows cytostatic action, prolongs the cell cycle S phase [3
], enlarges cell size [4
], and there is a clear anticarcinogenic activity of Resv in animal experiments [5
Previously, an increase in mitochondrial size by Resv has been observed in vivo [7
] and, in isolated rabbit renal tubules, experiments have demonstrated increased basal respiration following Resv exposure [9
]. On the other hand, experiments with isolated mitochondria show that Resv decreased the activity of several complexes in the electron transport chain (ETC) [10
] and directly interacted with complex I [12
]. Therefore, inhibition of the cellular respiration is suggested to be one of the primary targets of the cytotoxic effects of resveratrol [13
A proper cellular metabolism is a prerequisite for cellular proliferation, differentiation and apoptosis. Impaired mitochondria may alter cellular redox homeostasis, ATP production, the biosynthetic and secretory function of cells as well as nuclear gene expression by changing retrograde signaling pathways. Resv-induced mitochondrial biogenesis would act to overcome these impairments [7
]. When mitochondrial biogenesis is stimulated, as observed for Resv, a lower flow rate of electrons per unit of mitochondria is obtained, which decreases the propensity for free radical formation [15
]. Resv protects mitochondria against oxidative stress through AMP-Activated Protein Kinase-mediated Glycogen Synthase Kinase-3β inhibition downstream of the Poly (ADP-ribose) polymerase-LKB1 pathway [17
]. Since Rho 0 cells’ mitochondria are devoid of mtDNA and do not have oxidative phosphorylation, they can be used as a model for testing the molecular effects of lack of proper mitochondrial activity compared to a wild type (WT). The present experiments describe the effects of Resv on several mitochondrial activity parameters in HeLa WT and Rho0 cells. It is demonstrated that Resv treatment decreases cell numbers and proliferation, while an increase in cell size in HeLa WT is observed. Nevertheless, Resv increased OCR in both HeLa WT and Rho 0, whereas, for the cell line WT 143B and derived Rho0 cells, OCR was only altered by Resv in WT cells.
The mitochondrion is a central organelle for cellular proliferation and development and one of the proposed cellular targets for the effect of Resv. To analyze this in real time, a comparison of the effect of Resv on cells with and without functional mitochondria was made using the cervical cancer cell line HeLa with supporting data from 143B osteosarcoma cells.
The decreased cell proliferation rate in HeLa Rho 0 compared to HeLa WT is consistent with less ATP being produced via oxidative phosphorylation in the mitochondria, but data also showed (1) a higher mitochondrial mass; (2) increased expression of most of the tested mitochondria related mRNAs in HeLa Rho 0; and (3) an increased basal respiration in HeLa Rho 0. These features of HeLa Rho 0 cells indicate that the increased mitochondrial mass and TFAM expression could be a result from a compensatory survival strategy. In turn, a larger amount of mitochondria, which are dysfunctional, may result in an increased oxidative stress level. This is partly observed in HeLa Rho 0 cells when calculating the absolute values but not the oxidative stress level relative to the mitochondrial mass. Absence of the mitochondria encoded subunits prevents the assembly and function of mitochondrial respiratory complexes [18
]. However, HeLa Rho 0 mitochondria retain a proton gradient, which is essential for protein import into mitochondria and for counteracting apoptosis [19
]. This proton gradient is generated by hydrolyzing ATP imported from the cytoplasm by the ATP-ADP shuttle. ATP is hydrolyzed by the ATP synthase working in reverse mode to generate a transmembrane proton gradient [20
]. The enhanced expression of TFAM, Cyt c and most of the analyzed mitochondria related transcripts therefore indicates increased mitochondrial mass or volume in HeLa Rho 0.
The enhanced basal respiration in HeLa Rho 0 compared with HeLa WT can partially be explained by the increased non-mitochondrial respiration. In Rho 0 cells, NADH2
) is re-oxidized and electrons delivered to plasma-membrane oxidases, in order to recycle NAD+
for use in glycolysis [21
]. This flow of electrons will provide an increased non-mitochondrial OCR as seen in Figure 2
E. However, basal respiration in Rho 0 is increased above what can be explained by increased non-mitochondrial respiration, leaving some oxygen consumption unaccounted for. It should be noted, however, that substantial cell membrane oxygen consumption has been observed for different cell lines, including HelA subtypes [21
]. For comparison, 143B Rho 0 cells had a lower OCR (much lower than the 143B WT counterpart), which was unaffected by Resv treatment. To gain mechanistic insight into unaccounted non-mitochondrial respiration in HeLa Rho0 cells, these should be further studied using inhibitors, since it is evident from other studies that plasma membrane redox system is a Resv target [22
]. Moreover, when comparing the OCR/ECAR ratios of the Hela WT and HelA Rho 0 versus the 143B WT and 143B Rho 0, there were similar ratios for the two WT cell lines (4.8 ± 1.6) in contrast to a much higher ratio in HeLa Rho 0 cells (8.4 ± 1.0) and a much lower ratio for 143B Rho 0 cells (2.7 ± 0.9). This indicates different adaptive strategies for Rho 0 cells to obtain the necessary energy input.
Cells in general, and especially tumor cells, require a sufficient amount of ATP in order to synthesize bioactive compounds for rapid cell proliferation [23
] and different tumor cells use different strategies to gain sufficient amounts of ATP for the increased cell proliferation. Whereas some have increased glycolysis, others have an enhanced oxidative phosphorylation pathway [24
]. Our present data indicate that HeLa Rho 0 and WT have a different source of ATP production, due to the lack of well-functioning mitochondria, which also have an impact on regulation of glycolysis.
Numerous studies show a clear reduction in cell proliferation by Resv, which is confirmed by present data. Here, we significantly extend these findings by the observation that functional mitochondria are necessary for the cellular response to Resv and a Resv-induced cell enlargement. Previous findings show that Resv does not have an effect on cell doubling time in PC3 Rho 0 cells compared to the corresponding WT cells [25
]. Cell size is coupled to cell cycle progression and is affected by both internal and external cues. Cells must reach a certain size, before they progress in the cell cycle. Here is presented that Resv increases cell size in HeLa WT but not in the mitochondria deficient Rho 0 cells. In some cell types, Resv activates different pathways e.g., mechanistic target of rapamycin (mTOR) [26
], which could be the explanation to the increased cell size observed by Resv treatment in HeLa WT cells.
Resv increases basal OCR in both HeLa cell lines, but to a greater extent in HeLa WT, suggesting that mitochondrial function is indeed increased by Resv. On the other hand, the non-mitochondrial respiration is highly increased but only in HeLa Rho 0 cells, which indicates that Resv regulates a non-mitochondrial target as well. In contrast to the increased oxygen consumption rates, Resv did not increase ROS levels (Figure 3
B). Previous data reports that Resv increases the mitochondrial mass in HeLa WT cells [7
], which this experiment was unable to replicate.
Resv shows an increase in both mitochondrial oxidative as well as glycolytic activity for HeLa WT and Rho 0 (only significant for WT). Our data support the notion that HeLa WT has a propensity for using glycolysis for energy production, which underpins the importance of mitochondrial recycling of NADH (Figure 4
C). Catabolite repression is an important part of a cell’s global control system. It is assumed that interplay of Resv with membranes of wild type cells probably can cause a negative effect on the catalytic region of ETC proteins and F0
-ATP, which is the opposite for Rho 0 cells. It is also clear that this cell model has decreased oligomycin response and absent FCCP response (Figure 2
A,B and Figure 4
Previous reports showed an inhibition of ETC by Resv using isolated mitochondria [10
]. Such inhibition was not observed in the present experimental setup using intact cells. Even cells exposed to 10 µM Resv for 48 h showed the same OCR even though the cell number was decreased significantly, which indicates an increased ETC activity per cell.
Most of the mitochondria related mRNAs levels, except Sirt1 and PGC-1α, are increased in HeLa Rho 0 cells. Thus, there appears to be nuclear compensatory transcription in response to the dysfunctional mitochondria. The reference gene used for normalization showed similar Ct values between cell lines. Resv only has a very minor impact on the level of these mRNAs, and if so, at a lower concentration of Resv than the concentration which increases basal OCR (Figure 2
C). The minor effect of Resv on mRNA levels in treated HeLa WT could indicate that cellular modulation by Resv primarily acts at protein level [26
HeLa Rho 0 show more than expected oligomycin linked reduction of OCR. Both HeLa Rho 0 and WT do need a functional oligomycin-sensitive ATP synthase as discussed above, but it is more likely that the HeLa Rho 0 cells used may not have a completely malfunctioning electron transporting chain. Therefore, in order to confirm the current findings in HeLa cells, additional Rho 0 cell types should be investigated.
The described experiments showed cellular effects of Resv in the range of 5 to 50 µM after 24 h. According to Yang et al., more than 90% of the administrated Resv is degraded after 24 h in sodium bicarbonate containing media, and the actual concentrations of Resv are assumed to be lower than the initial dose [27
]. As Resv binds to proteins and lipid structures in the media, the high degree of degradation described by Yang et al. is likely an overestimation.
The partly lacking effect of Resv in HeLa Rho 0 could be explained by the fact that Resv inhibits ATP synthase. Recent experiments have shown that mitochondrial calcium overload and the apposite is triggered in HeLa cells and other cancer cell types [28
], which support the notion; functional mitochondria are important for the effect of Resv.
4. Materials and Methods
HeLa WT and Rho 0 derived from cervical cancer cells were a gift from Dr. Claus Desler, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark [29
]. Dulbecco’s modified Eagle’s medium (DMEM), Hank's Balanced Salt Solution (HBSS), Penicillin/streptomycin and uridine were obtained from Sigma-Aldrich (Broendby, Denmark). Fetal bovine serum (FBS) was from Merck Millipore (Hellerup, Denmark). Resveratrol was acquired from Fluxome Science (Stenloese, Denmark)—now Evolva (Reinach, Switzerland)—and dissolved in DMSO.
4.2. Cell Culture
HeLa WT and HeLa Rho 0 were grown in DMEM supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. HeLa Rho 0 medium was supplemented with 200 µM uridine. Cells were cultured in Thermo Fisher Scientific NuncTM microplates (Roskilde, Denmark).
4.3. Determination of Cell Number and Size
To determine cell number and diameter in HeLa WT and HeLa Rho 0, a Beckman Coulter, Z2 Coulter particle counter and size analyzer (Pasadena, CA, USA) was used [30
]. Particles with diameter size > 7 μm were included for data analysis. All cell counts were carried out in quadruplicate for each treatment with three independent experiments. The cells were treated with DMSO (as solvent) and 10–50 µM Resv for 24 and 48 h.
4.4. Impedance Readings
Evaluating the effects of Resv on cell proliferation was based on real-time cell-based electronic sensor technology iCELLigence System [31
] (ACEA Biosciences, Inv., San Diego, CA, USA). Measurements were performed according to manufacturer´s protocol. Briefly: in an E-plate L8 dish, each well was filled with 200 µL medium and placed in the iCELLigence station in a 37 °C incubator. Before seeding the cells into the E-Plate L8 (15.000 cells per well), a background measurement was done with media. The HeLa WT and HeLa Rho 0 were cultured in E-plates L8 in duplicates for 24 h before treatment with Resv was initiated. The impedance signal is expressed as the cell index at each time point per concentration of Resv. The cells were treated with DMSO (as solvent), 10 or 20 µM Resv for up to 110 h.
4.5. Respirometry in Intact Cells
The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements were made using Seahorse Bioscience XF-24 instrument (Seahorse Bioscience, Copenhagen, Denmark). HeLa WT and HeLa Rho 0 were grown to 80–90% confluence and then treated with 0, 10, 20 or 30 µM Resv 24 h prior to the day of the respirometry. On the day of the assay, the cell medium was removed from the well and running medium (XF-DMEM assay buffer, Seahorse Bioscience) was placed into the wells and warmed to 37 °C for at least 30 min without 5% CO2 atmosphere. The injection ports of the sensor cartridge were filled with Glucose/Sodium Pyruvate (25 mM/1 mM), Oligomycin (10 µM), FCCP (30 µM), and rotenone/antimycin A (20 µM for both) (Seahorse Bioscience). The measurement cycle consisted of a 3-min mix, a 3-min wait and a 3-min measurement. Eight basal rate measurements were followed by injections, and each injection was followed by three to four measurement cycles. Activity estimates for any given treatment were based on the rates from three wells to calculate the investigated parameters. Basal OCR represents the net sum of all processes in the cell capable of consuming O2 including mitochondria and other oxidases. Basal ECAR is established with the first measurements, as well as OCR. Non-mitochondrial respiration is estimated by average of OCR measurements after addition of antimycin A and rotenone. Proton leak is calculated as the first measurement after addition of oligomycin subtracted from non-mitochondrial respiration. The OCR-related ATP production is calculated as proton leak subtracted from basal OCR. Basal ECAR represent the net sum of predominantly measure of lactic acid formed during glycolytic energy metabolism. After addition of oligomycin, increased glycolytic rate in response to loss of mitochondrial related ATP production is calculated as the average of oligomycin stimulated rate of ECAR.
4.6. Detection of Mitochondrial Mass, Mitochondrial Membrane Potential and Oxidative Stress by Flow Cytometry
After 24 h of treatment with 0 or 20 µM Resv., HeLa WT and HeLa Rho 0 cells were detached and re-suspended in HBSS buffer (10 mM HEPES, 140 mM NaCl and 25 mM CaCl2, pH 7.4). Cells were incubated with Mitotracker green (MTG) (Invitrogen M7514, Thermo Fisher Scientific, Roskilde, Denmark) (0.01 µM), Tetramethylrhodamine methyl ester (TMRM) (Invitrogen T668) (0.01 µM) at room temperature (RT) or 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen C399) (0.1 µM) at 37 °C, for 30 min. Analysis was performed using flow cytometry (FACS Calibur, Becton-Dickinson, NJ, USA) and analyzed by BD CellQuest™ Pro Analysis software (version 4.0.2, Becton Dickinson, Khs. Lyngby, Denmark). Data were normalized to the HeLa WT solvent condition due to variation in absolute signal strength between experiments.
4.7. Quantitative Real Time RT-PCR Assay
Total RNA was extracted using TRI-Reagent (Sigma-Aldrich, Broendby, Denmark) according to manufacturer’s instructions. Concentration and purity of RNA were determined by NanoDrop spectrophotometer ND 1000 (Fisher Scientific, Roskilde, Denmark) and stored at −80 °C until cDNA synthesis. In addition, 1 µg of total RNA was reverse transcribed using a Superscript III Reverse transcription (Invitrogen) according to manufacturer’s instructions. The cDNA was subjected to real time PCR using QuantiTect SYBR® Green PCR Kit (Qiagen, Venlo, Netherlands). A reaction contained 500 nM of each forward and reverses primer, and 2 µL of 10 fold diluted cDNA with final reaction volume 10 µL. The reaction was initiated by 95 °C for 15 min. and 40 amplification cycles were carried out with 10 s denaturation at 95 °C and 60 s annealing/elongation at 60 °C. Subsequently, a melting curve was carried out. Gene expression was normalized to mean of RPL23a rRNA signals. Oligonucleotide sequences are available on request.
4.8. Statistical Analysis
All data are expressed as mean ± SEM and data are presented as a pool of replicate measurements from 2–4 independent experiments. Multiple groups were analyzed using one-way ANOVA, with Bonferroni post-test correction for multiple comparisons using GraphPad Prism (version 6.07, GrapPad, La Yolla, CA, USA). A t-test was used to analyze differences in mRNA levels. A probability of less than 0.05 was considered to indicate a significant difference.