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Triggering apoptotic death of human malignant melanoma a375.s2 cells by bufalin: involvement of caspase cascade-dependent and independent mitochondrial signaling pathways.

Hsiao YP, Yu CS, Yu CC, Yang JS, Chiang JH, Lu CC, Huang HY, Tang NY, Yang JH, Huang AC, Chung JG - Evid Based Complement Alternat Med (2012)

Bottom Line: The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis.Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner.Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨ(m) and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan.

ABSTRACT
Bufalin was obtained from the skin and parotid venom glands of toad and has been shown to induce cytotoxic effects in various types of cancer cell lines, but there is no report to show that whether bufalin affects human skin cancer cells. The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis. A375.S2 cells were treated with different concentrations of bufalin for a specific time period and investigated for effects on apoptotic analyses. Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner. Flow cytometric assays indicated that bufalin promoted ROS productions, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and nitric oxide (NO) formations in A375.S2 cells. Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨ(m) and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions. Based on those observations, we suggest that bufalin-triggered apoptosis in A375.S2 cells is correlated with extrinsic- and mitochondria-mediated multiple signal pathways.

No MeSH data available.


Related in: MedlinePlus

Bufalin affects cells' morphological changes and reduces percentage of viable A375.S2 cells. Cells were plated onto MEM + 10% FBS with various concentrations of bufalin for 24 or 48 h. (a) The morphological changes were examined and photographed under phase contrast microscope, and (b) the total percentages of viable cells were determined utilizing a PI-exclusion method and by flow cytometry as described in Section 2. Each point is mean ± SD of three experiments, *P < 0.05 was significantly different from the untreated control.
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fig1: Bufalin affects cells' morphological changes and reduces percentage of viable A375.S2 cells. Cells were plated onto MEM + 10% FBS with various concentrations of bufalin for 24 or 48 h. (a) The morphological changes were examined and photographed under phase contrast microscope, and (b) the total percentages of viable cells were determined utilizing a PI-exclusion method and by flow cytometry as described in Section 2. Each point is mean ± SD of three experiments, *P < 0.05 was significantly different from the untreated control.

Mentions: In order to investigate the biological effects of bufalin, A375.S2 cells were treated with various concentrations of bufalin for 24 and 48 h, and cell morphological changes and cell death were determined. The results are shown in Figures 1(a) and 1(b), which indicated that bufalin induced morphological changes (Figure 1(a)) and caused cell death in a concentration-dependent manner (Figure 1(b)). We found that the half maximal inhibitory concentration (IC50) is 450.38 nM in bufalin-treated A375.S2 cells at a 48 h incubation. Based on these observations, we selected the concentration of 450 nM bufalin, which is close to IC50, for further assessing whether the growth-inhibitory and cell death effects of bufalin are accompanied by its effect on apoptotic cell death.


Triggering apoptotic death of human malignant melanoma a375.s2 cells by bufalin: involvement of caspase cascade-dependent and independent mitochondrial signaling pathways.

Hsiao YP, Yu CS, Yu CC, Yang JS, Chiang JH, Lu CC, Huang HY, Tang NY, Yang JH, Huang AC, Chung JG - Evid Based Complement Alternat Med (2012)

Bufalin affects cells' morphological changes and reduces percentage of viable A375.S2 cells. Cells were plated onto MEM + 10% FBS with various concentrations of bufalin for 24 or 48 h. (a) The morphological changes were examined and photographed under phase contrast microscope, and (b) the total percentages of viable cells were determined utilizing a PI-exclusion method and by flow cytometry as described in Section 2. Each point is mean ± SD of three experiments, *P < 0.05 was significantly different from the untreated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376545&req=5

fig1: Bufalin affects cells' morphological changes and reduces percentage of viable A375.S2 cells. Cells were plated onto MEM + 10% FBS with various concentrations of bufalin for 24 or 48 h. (a) The morphological changes were examined and photographed under phase contrast microscope, and (b) the total percentages of viable cells were determined utilizing a PI-exclusion method and by flow cytometry as described in Section 2. Each point is mean ± SD of three experiments, *P < 0.05 was significantly different from the untreated control.
Mentions: In order to investigate the biological effects of bufalin, A375.S2 cells were treated with various concentrations of bufalin for 24 and 48 h, and cell morphological changes and cell death were determined. The results are shown in Figures 1(a) and 1(b), which indicated that bufalin induced morphological changes (Figure 1(a)) and caused cell death in a concentration-dependent manner (Figure 1(b)). We found that the half maximal inhibitory concentration (IC50) is 450.38 nM in bufalin-treated A375.S2 cells at a 48 h incubation. Based on these observations, we selected the concentration of 450 nM bufalin, which is close to IC50, for further assessing whether the growth-inhibitory and cell death effects of bufalin are accompanied by its effect on apoptotic cell death.

Bottom Line: The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis.Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner.Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨ(m) and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan.

ABSTRACT
Bufalin was obtained from the skin and parotid venom glands of toad and has been shown to induce cytotoxic effects in various types of cancer cell lines, but there is no report to show that whether bufalin affects human skin cancer cells. The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis. A375.S2 cells were treated with different concentrations of bufalin for a specific time period and investigated for effects on apoptotic analyses. Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner. Flow cytometric assays indicated that bufalin promoted ROS productions, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and nitric oxide (NO) formations in A375.S2 cells. Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨ(m) and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions. Based on those observations, we suggest that bufalin-triggered apoptosis in A375.S2 cells is correlated with extrinsic- and mitochondria-mediated multiple signal pathways.

No MeSH data available.


Related in: MedlinePlus