Antiproliferative and cytotoxic effects of resveratrol in mitochondria-mediated apoptosis in rat b103 neuroblastoma cells

Abstract

Resveratrol, a natural compound, has been shown to possess anti-cancer, anti-aging, anti-inflammatory, anti-microbial, and neuroprotective activities. In this study, we examined the antiproliferative and cytotoxicity properties of resveratrol in Rat B103 neuroblastoma cells; although it's molecular mechanisms for the biological effects are not fully defined. Here, we examined the cellular cytotoxicity of resveratrol by cell viability assay, antiproliferation by BrdU assay, DNA fragmentation by DNA ladder assay, activation of caspases and Bcl-2 family proteins were detected by western blot analyses. The results of our investigation suggest that resveratrol increased cellular cytotoxicity of Rat B103 neuroblastoma cells in a dose-and time-dependent manner with IC(50) of 17.86 µM at 48 h. On the other hand, incubation of neuroblastoma cells with resveratrol resulted in S-phase cell cycle arrests which dose-dependently and significantly reduced BrdU positive cells through the downregulation of cyclin D1 protein. In addition, resveratrol dose-dependently and significantly downregulated the expression of anti-apoptotic protein includes Bcl-2, Bcl-xL and Mcl-1 and also activates cleavage caspase-9 and-3 via the downregulation of procaspase-9 and -3 in a dose-dependent manner which indicates that involvement of intrinsic mitochondria-mediated apoptotic pathway. In conclusion, resveratrol increases cellular cytotoxicity and inhibits the proliferation of B103 neuroblastoma cells by inducing mitochondria-mediated intrinsic caspase dependent pathway which suggests this natural compound could be used as therapeutic purposes for neuroblastoma malignancies.

Keywords: Apoptosis; Bcl-2 family; Cyclin D1; Neuroblastoma; Resveratrol.

Fig. 1

Resveratrol increases cellular cytotoxicity of Rat B103 neuroblastoma cells. (A, B) B103 cells were cultured in 96-well culture dishes to near confluence 50~60% and then starved in DMEM containing 0.5% FBS for 24 h. Cell death was determined by using the cytotoxicity assay kit (CCK-8, Dojindo Lab). Cells were exposed to resveratrol in a different dose of 0 to 20 µM in dose dependent and 15 µM in time dependent experiments. Each point is mean±SEM of quintuple samples. Data are mean from three independent experiments in which the activity in the absence of resveratrol versus in the presence of resveratrol is significantly different (n=3, ^**p<0.01). (C) B103 cells were grown in 24-well culture dishes to near confluence 50% and then starved in DMEM containing 0.5% FBS for 24 h. They were then added 15 µM concentration of resveratrol and grown at 37℃, in humidified 5% CO₂ for 48 h and then morphology was observed by Bright-Field Microscopy. Arrows indicate cells with apoptotic morphology. (D) B103 cells were grown in 100 mm culture dishes to near confluence 90% and then starved in DMEM containing 0.5% FBS for 24 h. The cells were then treated with 0, 15 and 20 µM of resveratrol. After 48 h resveratrol treatment, DNA was extracted and separated on 0.8% agarose gel containing ethidium bromide. DNA fragments were visualized under UV light. M indicates as a Marker. GA (Gambogic acid) used as a positive control.

Fig. 2

Antiproliferative effects resveratrol in DNA synthesis and cyclin D1 downregulation. (A) B103 cells were culture on a round cover slides in 24 well dishes until 80% confluence in 10% FBS. The cells were then treated in the absence and presence of 15 µM of resveratrol. After 46 h resveratrol treatment, cells were incubated with 10 µM BrdU for 2 h before fixation and subsequent immunostaining with an anti-BrdU antibody (Arrow shows BrdU positive cells). (B) The percentages of stained cells were counted in three independent random areas. Results are means±SE and representatives of three independent experiments are shown (n=3, ^*p<0.05). (C) B103 cells were cultured in 60-mm culture dishes to near 80% confluence in 10% FBS. They were treated with 0 to 20 µM of resveratrol for 48 h. Equal volumes of whole-cell extracts containing 40 µg of protein were separated and electrophoretically blotted. Cyclin D1 was detected via immunoblot analysis. β-actin was used as a loading control. (D) The intensities of the cyclin D1 bands were determined by densitometric scanning and analyzed by Bio-Profil software and the expression levels were normalized to β-actin. Results are mean±S.E. and representatives of three independent experiments are shown (n=3, ^*p<0.05, ^**p<0.01).

Fig. 3

Resveratrol down-regulates Bcl-2 family proteins. (A) B103 cells were cultured in 60-mm culture dishes to near 90% confluence and then starved in DMEM containing 0.5% FBS for 24 h. After 24 h starvation cells were treated with 0 to 20 µM of resveratrol. Whole cell lysates were subjected to 15% SDS-PAGE and the levels of Bcl-2, Bcl-xL, and Mcl-1 were detected by western blotting as described in materials and methods. β-actin was used as a loading control. (B~D) The intensities of the Bcl-2 bands, the Bcl-xL bands and the Mcl-1 bands were determined by densitometric scanning and analyzed by Bio-Profil software and the expression levels were normalized to β-actin. Results are mean±S.E. and representatives of three independent experiments are shown (n=3, ^*p<0.05, ^**p<0.01, ^***p<0.001).

Fig. 4

Effects of resveratrol in caspase expression. (A) B103 cells were cultured in 60-mm culture dishes to near 90% confluence and then starved in DMEM containing 0.5% FBS for 24 h. They were treated with 0 to 20 µM of resveratrol for 48 h. Levels of caspase-9 and -3 proteins were detected by western blotting. β-actin was used as a loading control. (B, C) The intensities of the cleaved caspase-9 bands and the cleaved caspase-3 bands were determined by densitometric scanning and analyzed by Bio-Profil software and the expression levels were normalized to β-actin. Results are mean±S.E. and representatives of three independent experiments are shown (n=3, ^*p<0.05, ^**p<0.01). (D) B103 cells were cultured in 96-well dishes to near confluence 50~60% and then starved in DMEM containing 0.5% FBS for 24 h. After starvation cells were pretreated with 10 µM DEVD-FMK for 1 h just before exposure to 15 µM resveratrol for 48 h. Cell number was quantified by CCK-8 kit. Results are means±SE and representatives of three independent experiments are shown (n=3, ^*p<0.05).