Figure 1.
Chemical structure of the mitochondria-targeted curcuminoids used in this study.
(A) Curcumin; (B) Mitocur-1; (C) Mitocur-2; (D) Mitocur-3. The synthetic curcuminoids (Mitocur-1,2,3) have a triphenylphosphonium cation linked to the hydroxyl group(s) of curcumin via a three-carbon propyl chain. Mitocur-3 is similar to Mitocur-1, but is devoid of methoxy substitution on the aryl groups.
Table 1.
Cellular uptake of curcumin and mitocurcuminoids-1, 2, and 3.
Table 2.
Curcumin and Mitocur-1, 2 and 3 induce cell death in various cancer cells by SRB Assay.
Figure 2.
Mitocur-1, 2 and 3 induces superoxide generation in MCF-7 cells.
(A) Ethidium fluorescence of MCF-7 cells treated with curcumin (50 µM) and mitocur-1, 2, 3 (10 µM) for 4 h. All experiments were performed with and without NAC (4 mM). Representative fluorescence images are shown. (B) shows quantification of fluorescence intensities shown in A. **, significantly different compared to control (P<0.01) and #, significantly different compared to mitocurcuminoid-1, 2 and 3 (P<0.01). (C) cells were treated with or without 10 µM mitocurcuminoid-1 and 4 mM NAC for 24 h. Results are expressed percentage of viable cells compared to control. Data represented is from at least three independent experiments. **, significantly different compared to control (P<0.01) and #, significantly different compared to NAC (P<0.01).
Figure 3.
Superoxide generation in MCF-7 cells treated with mitocur-1 detected by EPR spectroscopy using DMPO spin trap.
(A) Cells alone; (B) Cells+curcumin (10 µM); (C) Cells+mitocur-1Mitocur- (10 µM); (D) Cells+mMitocur-1 (10 µM)+MnTBAP (10 µM); (E) xanthine+xanthine oxidase. Microwave frequency, 9.786 GHz; microwave power, 10 mW; modulation amplitude, 1 G; scan time, 30 s; no. of scans, 10.
Figure 4.
Effect of mitocurcuminoids and curcumin on mitochondrial membrane potential and apoptotic markers.
(A) Cells were treated with 10 µM Mitocur-1, 2, 3 or 50 µM curcumin for 4 h. Then washed with PBS and incubated with JC-1 dye (5 µg/ml) for 20 min to measure the loss of mitochondrial membrane potential. Fluorescence images were captured in both FITC and rhodamine filters and images showing J-aggregates are represented. (B) shows quantification of images (J-aggregates) shown in A. (C) Mitochondria and cytosolic fractions were isolated using ProteoExtract Cytosol/Mitochondria Fractionation Kit and cytochrome c levels were measured by Western blot analysis. (D) MCF-7 cells were treated with Mitocur-1 (5 and 10 µM) for 24 h. Total protein was resolved by SDS-PAGE electrophoresis and Western blot analysis was performed using respective antibodies for Bcl2, Bax, caspase-7 and PARP. **, significantly different compared to control (p<0.01).
Figure 5.
Modulation of cell cycle progression by Mitocur-1.
MCF-7 cells were treated with Mitocur-1 (5 and 10 µM) for a period of 24 h. (A) shows the flow cytometry profiles of (PI)- stained cells of control, and Mitocur-1 (5 and 10 µM) treatment as described in Methods. (B) Quantitative cell cycle (DNA content) distribution (% of total) in the control and treatment groups. (C) MCF-7 cells were treated with Mitocur-1 (5 and 10 µM) for 24 h and subjected to Western blot analysis. Representative immunoblot images of cyclin A, cyclin B1 and cyclin D1 are shown. Values are expressed Mean ± SD; (n = 4). **, significantly different from control (P<0.01).
Table 3.
Mitocur-1 increase caspase 3-like and caspase-8 activities in MCF-7 cells.
Figure 6.
Mitocur-1 causes inhibition of Akt and STAT3 signaling and increase the ERK1/2 phosphorylation.
MCF-7 cells were treated with Mitocur-1 (5 or 10 µM) for 16 h and subjected to Western blot analysis as described in Methods. Representative immunoblot images of STAT3, phosphorylated STAT3 (Tyr705), Akt, phospho-Akt (Thr-308), ERK1/2 and phospho- ERK1/2 are shown.
Figure 7.
Curcumin and Mitocur-1 presumably regulates BNIP3 expression by modulating DNMT1 but not through HDAC's.
(A) MCF-7 cells were treated with various concentrations of either mitocurcuminoid-1 (1–10 µM) or curcumin (5–50 µM) for a period of 24 h. Total RNA was extracted by TRIzol method and RT-PCR was performed using gene specific primers of BNIP3. (B) and (C) are same as A, except that cells were treated with AZA, (24 µM) or TSA (200 nM). Relative quantifications of BNIP3 expressions were presented in bar graphs. *, significantly different compared to control (p<0.01).