Korean J Urol.  2010 Dec;51(12):870-878.

Dimethoxycurcumin, a Structural Analogue of Curcumin, Induces Apoptosis in Human Renal Carcinoma Caki Cells Through the Production of Reactive Oxygen Species, the Release of Cytochrome c, and the Activation of Caspase-3

• Lee JW
• Hong HM
• Kwon DD
• Pae HO
• Jeong HJ

Abstract

PURPOSE
Curcumin (Cur) has been reported to induce apoptosis in human renal carcinoma Caki cells. Dimethoxycurcumin (DMC), one of several synthetic Cur analogues, has been reported to have increased metabolic stability over Cur. We determined whether DMC, like Cur, induces apoptosis in Caki cells and also compared the apoptosis-inducing activity of DMC with that of Cur.
MATERIALS AND METHODS
Caki cells were treated with DMC possessing four methoxy groups, Cur possessing two methoxy groups, or bis-demethoxycurcumin (BMC), which lacks a methoxy group. Cell viability was measured by using a methyltetrazolium assay. Flow cytometry and the caspase-3 activity assay were used to detect apoptosis. The release of cytochrome-c (Cyt c) was detected by Western blot analysis. The production of reactive oxygen species (ROS) was measured by flow cytometry.
RESULTS
DMC, Cur, and BMC reduced cell viability and induced apoptosis, but the potency varied; DMC was the most potent compound, followed by Cur and BMC. ROS production, Cyt c release, and caspase-3 activity were increased, again in the order DMC>Cur>BMC. N-Acetylcysteine, a potent antioxidant, inhibited ROS production, Cyt c release, caspase-3 activation, and apoptosis induction in DMC-treated cells.
CONCLUSIONS
These results indicate that DMC, like the original form of Cur, may induce apoptosis in human renal carcinoma Caki cells through the production of ROS, the release of mitochondrial Cyt c, and the subsequent activation of caspase-3. In addition, DMC is more potent than Cur in the ability to induce apoptosis.

Keyword

Antineoplastic agents; Apoptosis; Curcumin; Dimethoxycurcumin; Renal cell carcinoma

MeSH Terms

Acetylcysteine
Antineoplastic Agents
Apoptosis
Blotting, Western
Carcinoma, Renal Cell
Caspase 3
Cell Survival
Chlorobenzenes
Curcumin
Cytochromes
Cytochromes c
Flow Cytometry
Humans
Reactive Oxygen Species
Acetylcysteine
Antineoplastic Agents
Caspase 3
Chlorobenzenes
Curcumin
Cytochromes
Cytochromes c
Reactive Oxygen Species

Figure

• 

  FIG. 1 Chemical structures of dimethoxycurcumin (DMC), curcumin (Cur), and bis-demethoxycurcumin (BMC). Note that the original form of Cur contains two methoxy groups at two aromatic rings, whereas DMC contains four, and BMC contains none.

• 

  FIG. 2 Effects of dimethoxycurcumin (DMC), curcumin (Cur), and bis-demethoxycurcumin (BMC) on viability and DNA fragmentation of Caki cells. (A) Cells were treated for 24 h with the indicated concentrations of DMC. (B) Cells were treated with 80 µM of DMC for the indicated times. (C) Cells were treated for 24 h with 80 µM of DMC, Cur, or BMC. Cell viability was analyzed by use of the MTT assay. Each bar represents the Mean±SD (n=4). a: p<0.05 compared with the control groups, b: p<0.05 compared with the DMC-treated groups. (D) Cells were treated for 12 h with 10 µM of H2O2 (served as a positive control) or for 24 h with 80 µM of DMC, Cur, or BMC, and DNA fragmentation was analyzed by agarose gel electrophoresis. This experiment was repeated three times with similar results.

• 

  FIG. 3 Effects of dimethoxycurcumin (DMC), curcumin (Cur), and bis-demethoxycurcumin (BMC) on apoptosis in Caki cells. (A) Cells were treated for 12 h with solvent (control) or with 80 µM of DMC and were then stained by Annexin V-FITC/PI solution and analyzed by flow cytometry (cells in the lower right quadrant represent apoptosis). Cells were treated for 12 h with the indicated concentrations of DMC (B) or with 80 µM of DMC, Cur, or BMC (C). After Annexin V/PI staining, flow cytometric analysis was performed to determine the percentage of Annexin-V positive cells. Each bar represents the Mean±SD (n=4). a: p<0.05 compared with the control groups, b: p<0.05 compared with the DMC-treated groups.

• 

  FIG. 4 Effects of dimethoxycurcumin (DMC), curcumin (Cur), and bis-demethoxycurcumin (BMC) on reactive oxygen species (ROS) production in Caki cells. (A) Cells stained by DCF-DA were treated for 6 h with solvent (control) or with 80 µM of DMC and were then analyzed by flow cytometry. Data are presented as log fluorescence intensity. Cells stained by DCF-DA were treated for 6 h with the indicated concentrations of DMC (B) or with 80 µM of DMC, Cur, or BMC (C). Flow cytometric analysis was performed to determine the mean fluorescence intensity of DCF-positive cells. Each bar represents the mean±SD (n=4). a: p<0.05 compared with the control groups, b: p<0.05 compared with the DMC-treated groups.

• 

  FIG. 5 Effects of dimethoxycurcumin (DMC), curcumin (Cur), and bis-demethoxycurcumin (BMC) on cytochrome c (Cyt c) release and caspase-3 activity in Caki cells. Cells were treated for 6 h with the indicated concentration of DMC (A) or with 80 µM of DMC, Cur, or BMC (B). Subcellular fractions were prepared and cytosolic Cyt c was detected by Western blot analysis with Cyt c antibody. These experiments were repeated three times with similar results. Cells were treated for 12 h with the indicated concentrations of DMC (C) or with 80 µM of DMC, Cur, or BMC (D). Caspase-3 activity was analyzed by a fluorogenic assay, as described in the Materials and Methods. Each bar represents the mean±SD (n=4). a: p<0.05 compared with the control groups, b: p<0.05 compared with the DMC-t reated groups.

• 

  FIG. 6 Effects of N-acetylsysteine (NAC) on reactive oxygen species (ROS) production, cytochrome c (Cyt c) release, caspase-3 activation, and apoptosis induction in dimerthoxycurcumin (DMC)-treated Caki cells. Cells were pre-incubated for 1 h with 10 mM of NAC and were then exposed for either 6 h (A, B) or 12 h (C, D) to 80 µM of DMC. ROS production (A) and apoptosis induction (D) were determined by flow cytometric analysis using DCF-DA and Anennxin V plus PI, respectively. Cytosolic Cyt c (B) was detected by Western blot analysis using Cyt c antibody. Caspase-3 activity (C) was analyzed by a fluorogenic assay. Each bar represents the Mean±SD (n=4). a: p<0.05 compared with the control groups, b: p<0.05 compared with the groups treated with DMC alone.

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