Mitochondrial membrane potential assay
Determination of the mitochondrial transmembrane potential (Dym) was performed using 5,5,6,-tetrachloro-1,1,3,3tetraethylbenzimidazolyl carbocyanine iodide (JC-1) assay, as previously described.14 KKU-M156 cells were seeded onto 96 black-well plate with density of 2x105 cells/well and incubated overnight for adherence. The media was then replaced with fresh media containing phenformin, quercetin 20 µM, and cyclosporine 50 nM and incubated for 6 h. The culture media was replaced with 5 µM JC-1 staining solution in serum free media (dilution 1:20) and incubated at 37oC for 45 min. The cells were washed with JC-1 buffer and measured fluorescent signals using a fluorescent microplate reader. The excitation and emission wavelengths were set at 485/535 nm (cut-off 515 nm) for measurement of J-monomers and at 535/595 nm (cut-off 590) for J-aggregates.
Statistical analysis
All values were presented as mean ± SEM from three experiments using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA, USA). Statistical differences between treatment and control groups were analyzed by one-way analysis of variance (ANOVA) with post-hoc test or Students t-test by SigmaStat version 3.11 software (Systat Software, San Jose, CA, USA). Statistical significance was preset at p<0.05.
Results
Antitumor activity of phenformin and flavonol compounds
The structure of phenformin, quercetin and myricetin were shown in Fig.1A. Phenformin was tested for cytotoxicity in KKU-156 cells. It was shown that phenformin induced cytotoxicity in concentration-response relation with median inhibitory concentration (IC50) of 1363±427 µM (Fig.2A). When phenformin was used in combination with quercetin or myricetin at varied concentrations, the cytotoxicity was significantly increased in co-treatment with quercetin, when compared with phenformin alone. But the combination of phenformin and myricetin did not increase cytotoxicity (Fig. 2B and C). Quercetin alone at the same concentration used in the combinations induced cytotoxicity by 27.5±3.6%, while myricetin showed cytotoxic effect by 14.8±2.4%. It is apparent that quercetin has an additive cytotoxic effect to phenformin.
Figure 1 The structure of (A) phenformin, (B) quercetin, and (C) myricetin
Figure 2 Antiproliferative effect of phenformin in CCA cells. KKU-M156 cells were treated with various concentrations of (A) phenformin (Phen), (B) Phen in combinations with quercetin (QC) or with myricetin (MR) and incubated for 24 h. After treatment, the cell viability was determined using the SRB assay. Each bar represents the mean ± SEM of three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 Phen alone versus the corresponding combination of Phen and QC.
Phenformin and quercetin induced mitochondrial dysfunction
As mitochondria are the powerhouse of energy production by oxidative phosphorylation in mitochondrial matrix and play central role in cell death, disturbance in mitochondrial function may lead to cell dysfunction and cell death.15 Phenformin induced the loss of mitochondria transmembrane potential Dym), as determined by JC-1 assay. In healthy mitochondria, JC-1 is taken up into the mitochondria and forming J-aggregates which emit the red-orange fluorescent signal. The damage mitochondria cannot accumulate JC-1, then JC-1 exists as J-monomer form in cytosol and emits the green fluorescent signal. The ratio of red and green fluorescent signals is an indicative of the status of Dym. It was apparent that phenformin induced decline of the ratio of J-aggregates/J-monomers in concentration response manner (Fig.3A and B). The loss of Dym, was even exaggerated in the presence of quercetin. It is noted that quercetin itself also induced the loss of Dym,.
To examine whether the change in Dym, was due to the opening of the mitochondrial permeability transition pore (MPTP), which plays critical role in maintenance of mitochondrial transmembrane potential. Cyclosporine A, a MPTP inhibitor, was used in the study. Cyclosporine could partial prevent the loss of ym, induced by phenformin or quercetin or the combinations of phenformin and quercetin (Fig. 4 and 5A, B). This indicates that the depolarization of mitochondria is due to the opening of MPTP.
Figure 3 Mitochondrial transmembrane potential (ym) in CCA cells treated with phenformin and quercetin. KKU-M156 cells were treated with phenformin (Phen) and combination with quercetin (QC) for 6 h. After treatment, ym was determined by JC-1 assay. (A) Representatives of images of KKU-M156 treated with Phen and QC. (B) Bar graph of fluorescent signal of red/green ratio. Each bar represent the mean ± SEM from three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 Phen alone versus the combination of Phen and QC.
Figure 4 Mitochondrial transmembrane potential (ym) in cells treated with phenformin and cyclosporine. KKU-M156 cells were treated with phenformin (Phen) and cyclosporine (CS) for 6 h. After treatment. ym was determined using the JC-1 assay. (A) Representatives of images of KKU-M156 after treatment. (B) Bar graph of fluorescent signal of red/green ratio. Each bar represents the mean ± SEM from three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 Phen alone versus the combination of Phen and QS.
Figure 5 Mitochondrial transmembrane potential (ym) in cells treated with phenformin, quercetin and cyclosporine. KKU-M156 cells were treated with phenformin (Phen), quercetin (QC), and cyclosporine (CS) for 6 h. After treatment, ym was measured by the JC-1 assay. (A) Representatives image of KKU-M156 after treatment. (B) Bar graph of fluorescent signal of red/green ratio. Each bar represents the mean ± SEM from three independent experiments. *p < 0.05 versus untreated control group, #p < 0.05 combination of Phen and QC versus the combination of Phen, QC and QS.
Discussion
Phenformin inhibits several cancer growth including some resistant cancer cells.13,16 The mechanism of action is suggested to be mediated via AMPK-mTOR in the suppression of anabolic process and cause oxidative stress through mitochondrial pathway.13,16 Quercetin and myricetin both potently inhibit glucose transport in rat adipocytes.12 It was then suggested that interference of glucose uptake by these flavonols could enhance phenformin in suppression of tumor cell growth, since tumor cells generally requires high glucose for their proliferation and energy metabolism.17 However, only quercetin shows enhanced cytotoxicity of phenformin in CCA cells. Differential effect between quercetin and myricetin may be that quercetin, but not myricetin could affect glucose uptake in HepG2 cells.18 It is probable that effects on glucose transport is cell type-dependent and needs further investigation. Moreover quercetin is among flavonoid derivatives which show to be a potent AMPK activators and could suppress mTOR pathway.10
The present study showed that only quercetin significantly enhances the cytotoxicity of phenformin. The inhibition of glucose transport may be one among many other effects which mediate antiproliferation of cancer cells. Phenformin causes the loss of Dym, which is due to the opening MPTP and that leads to the outer mitochondrial membrane permeabilization and consequence to cell death.15 The effect via mitochondrial is validated by using cyclosporine, a MPTP inhibitor.19 Cyclosporine can partially inhibit the loss of Dym. The effect of phenformin on mitochondrial function is shown to be similar to metformin which leads to oxidative stress and cell death.20 Moreover, quercetin alone induces the loss of Dym, which the effect is validated by cyclosporine. The enhanced antitumor effect of quercetin may be by enhanced dysfunction of mitochondria with consequence of increased cell death.
It is concluded that quercetin increases phenformin-induced cell death by intensification of mitochondrial damage which leads to cell death. Quercetin in combination with phenformin increase efficacy of antitumor activity and could be a possible effective strategy in cancer chemoprevention.
Acknowledgements
This study was granted by Faculty of Medicine, Khon Kaen University, Thailand (Grant Number IN62302).
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