Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells

<div><p>In our previous study, we found that pretreatment with lipoamide (LM) more effectively than alpha-lipoic acid (LA) protected retinal pigment epithelial (RPE) cells from the acrolein-induced damage. However, the reasons and mechanisms for the greater effect of LM than LA are unclear. We hypothesize that LM, rather than the more direct antioxidant LA, may act more as an indirect antioxidant. In the present study, we treated ARPE-19 cells with LA and LM and compared their effects on activation of mitochondrial biogenesis and induction of phase II enzyme systems. It is found that LM is more effective than LA on increasing mitochondrial biogenesis and inducing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its translocation to the nucleus, leading to an increase in expression or activity of phase II antioxidant enzymes (NQO-1, GST, GCL, catalase and Cu/Zn SOD). Further study demonstrated that mitochondrial biogenesis and phase II enzyme induction are closely coupled via energy requirements. These results suggest that LM, compared with the direct antioxidant LA, plays its protective effect on oxidative damage more as an indirect antioxidant to simultaneously stimulate mitochondrial biogenesis and induction of phase II antioxidant enzymes.</p></div

LM increased ETC complex I, II, III, IV and V protein expression (A-E), mitochondrial DNA copy number (F) and viable mitochondria (G).

<p>ARPE-19 cells were treated with the indicated concentrations of LM for 48 h, and then subunits expression of the complexes were detected by western blot. The subunits tested were 39 KD, 70 KD, 51.6 KD, 57 KD and 56.6 KD for complexes I to V (A to E), respectively. The images are representative; the quantitative results are from optical density analysis of images of three independent experiments. For complexes I, II and V, the loading control was β-actin; for complexes III and IV, it was α-tubulin instead. Results are the ratios of the complex densities to those of β-actin or α-tubulin. Values are means ± SEM. Differences were evaluated statistically with student’s t test. * p<0.05, and **p<0.01 vs. untreated control (0 μmol/L). <b>(F)</b> LM increased viable mitochondria and mitochondrial DNA copy numbers. ARPE-19 cells were treated with the indicated concentrations of LM for 48 h. For viable mitochondria measurement, cells were stained with Mitotracker Green. Fluorescence values read by flow cytometry were considered as estimates of viable mitochondria. Results are in arbitrary units normalized by setting the fluorescence of untreated (0 μmol/L LM) cells to 100. Values are means ± SEM from three independent experiments, each performed on three samples at each concentration. For mitochondrial DNA copy number measurement, real-time PCR was employed for assaying the D-LOOP region of mitochondrial DNA. The results shown are ratios of D-LOOP to 18S rDNA. Results are in arbitrary units normalized by setting the ratio of untreated (0 μmol/L LM) cells to 100. Values are means ± SEM from four independent experiments. Statistical significance of differences was established by student’s t test. * p<0.05, vs. 0 μmol/L treatment and **p<0.01 vs. untreated control (0 μmol/L) <b>(G)</b> A representative flow cytometry histogram was created with Flow Jo, Ver. 4.87 software. The fluorescence curves of 0, 10 and 40 μmol/L LM treatments were right-shifted with respect to the 0 μmol/L curve.</p

LM treatment increased Nrf2 expression in both total and nuclear protein fraction, and increased expression and activity of NQO-1.

<p>ARPE-19 cells were treated with the indicated concentrations of LM for 48 h, or 40 μmol/L of LA or LM if concentrations not indicated. <b>(A)</b> A representative image of Nrf2 expression in total protein detected by western blot. Optical densities were analyzed with Quantity One software, and results are expressed as ratios of Nrf2 to β-actin in arbitrary units. <b>(B)</b> A representative image of Nrf2 expression in nuclear protein. Quantitative analysis of Nrf2 expression in nuclear protein was quantified in the same way as for Nrf2 expression in total protein. <b>(C)</b> A representative image of NQO-1 expression detected by western blot. Quantitative analysis of NQO-1 expression in total protein was performed in the same wa with Nrf2.. <b>(D)</b> A representative image of NQO-1 expression. <b>(E)</b> Quantitative analysis of NQO-1 expression and activity. All values are means ± SEM of four independent experiments. Statistical significance was established by one way ANOVA followed by the Tukey test. *p<0.05, and **p<0.01 vs. control, and ^#p<0.05, ^##p<0.01 vs. LA treatment.</p

Primers and annealing temperature.

<p>Primers and annealing temperature.</p

The effects of LM on oxygen consumption (A), mitochondrial membrane potential (MMP) (B), ROS production (C); cellular ATP level (D) and the expression of MnSOD,Trx2,Prx3,and Prx5 (E).

<p>ARPE-19 cells were treated with 40 μmol/L LM for 48 hours; then the following assays were carried out immediately. <b>(A)</b> LM promoted oxygen consumption. Results are expressed as the rate of oxygen consumption, with media without cells used as a blank. Values are means ± SEM from three independent experiments; three parallel measurements were used for each sample in every experiment. <b>(B)</b> LM treatment increased MMP as determined by JC-1 staining. Values are means ± SEM of the ratio of fluorescence at 590 nm to 530 nm from three independent experiments; 4 parallel wells for each group were used in each experiment. <b>(C)</b> LM treatment decreased ROS production examined by DCF-DA staining. Values are means ± SEM of 8 parallel wells of a representative experiment, from four independent experiments each showing similar trends. (D) LM treatment decreased cellular ATP level. Values are means ± SEM from 3 independent experiments. (E) Expression of MnSOD,Trx2,Prx3,and Prx5. ARPE-19 cells were treated with 40 μmol/L LM or LA for 48 h; then RNA was isolated and reverse-transcribed to cDNA. Real time PCR was employed to measure expression levels of the indicated genes. The results (from 5 independent experiments) are expression ratios of the target genes to 18SrRNA, and are normalized to control (control = 100). C stands for control, LM stands for 40 μmol/L LM treatment and LA stands for 40 μmol/L LA treatment. Statistical significance was established by one way ANOVA followed by the Tukey test (A, B, C, D) or LSD test (E). * p<0.05, ** p<0.01 vs. untreated control (0 μmol/L); ^#p<0.05, ^## p<0.01 vs. LA.</p

The effects of LM and LA on expression and activity of GST, catalase, Cu/ZnSOD and G6PD.

<p>ARPE-19 cells were treated with 40 μmol/L LM or LA for 48 h. <b>(A)</b> A representative image of total GST expression detected by western blot. <b>(B)</b> Quantitative analysis of GST expression (4 independent experiments) and activity (5 independent experiments). <b>(C)</b> A representative image of catalase expression detected by western blot. <b>(D)</b> Quantitative analysis of catalase expression (4 independent experiments) and activity (3 independent experiments). The results are expressed in arbitrary units and each experiment was performed in duplicate. <b>(E)</b> Transcriptional expression of Cu/ZnSOD. Real time RT-PCR was employed to measure expression levels of Cu/ZnSOD. Results (from 5 independent experiments) are expressed as ratios of Cu/ZnSOD to 18SrRNA. <b>(F)</b> G6PD activity was measured as described in Methods. Values are means ± SEM of three independent experiments. All statistical significance were established by one way ANOVA followed by the Tukey test. * p<0.05, **p<0.01 vs. untreated controls (0 μmol/L), ^#p<0.05 vs. LA, and ^##p<0.01 vs. LA.</p

FKB mediated-AR transcriptional repression requires the expression of Sp1.

<p>(<b>A</b>).C4-2B cells were co-transfected with PSA-Luc or plARS-Luc, along with a Renilla luciferase plasmid pGL 4.71 and Luciferase activities were measured. FKB significantly decreases promoter activities of <i>AR</i> and <i>PSA</i> genes (Student <i>t</i> test, P<0.01). Bars are mean ± SD of three independent experiments. (<b>B</b>).Western blotting analysis of Sp1 protein expression. A representative blot was shown from three independent experiments. α -Tubulin was detected as a loading control. (<b>C</b>). ChIP analysis of the binding of Sp1 to the AR promoter sequence reveals that 16 hours FKB treatment results in a significant decrease in the binding of Sp1 to the AR promoter sequence that contain two Sp1 binding sites. Mouse IgG was used as a blank control. (<b>D</b>). Overexpression of Sp1 in C4-2B cells attenuates FKB induced AR protein down-regulation. After 48 hours of Sp1-HA plasmid transfection, C4-2B cells were treated with FKB for 16 hours. Sp1 and AR protein levels were examined. A representative blot was shown from three independent experiments. α -Tubulin was detected as a loading control.</p

FKB and yangonin or 5′6;-dehydrokawain act synergistically to reduce the growth of C4-2B cells and down-regulate the expression of the full-length AR and an AR splice variant.

<p>The growth inhibitory of FKB and yagonin (<b>A</b>), or 5′6;-dehydrokawain (<b>B</b>), or kawain (<b>C</b>), or methysticin (<b>D</b>) alone and in combination on C4-2B cells. Each point is the mean of four independent experiments; bars, SD. Each sample was counted in duplicate. IC_{50 s} were estimated by dose-response curves, IC_{50 s} of the combinations are significantly lower than those of the treatments alone (Student <i>t</i> test, P<0.01). (<b>E</b>) <b>and</b> (<b>F</b>) Western blotting analysis of expression of the full-length AR (110 KDa), the AR splice variant (83 KDa) and PSA in C4-2B and 22Rv1 cells, respectively. A representative blot was shown from three independent experiments. α -Tubulin was detected as a loading control.</p

The IC_{50 s} of the kava root extract and its active components and AR status in prostate cell lines.

<p>*AR status: MT: mutant; SP: splicing variant; WT: wild-type; ND: not detectable.</p>#<p>Androgen dependency: AD: androgen is required for growth; AS: cells may respond to androgen, but do not require it for growth; AI: cells do not respond to androgen.</p

The effect of the kava root extract and its active components on the growth of PCa cell lines (C4-2B, LNCaP, 22Rv1, LAPC-4, DU145 and PC3) and a prostate myofibroblast WPMY-1.

<p>Cells in 24-well culture plates were treated with 0.1% DMSO, the kava root extract, kawain, 5′, 6′-dehydrokawain, yangonin, methysticin, or flavokawain B (FKB) at the indicated doses. After 72 hours of treatment, cell densities were measured by MTT assay. Each point is the mean of values from four independent plates; bars, SD. Each sample was counted in duplicate. IC_{50 s} were estimated by dose-response curves.</p