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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

A schematic diagram showing the proposed signaling pathways of bufalin-induced apoptosis in A375.S2 human malignant melanoma cells.
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fig5: A schematic diagram showing the proposed signaling pathways of bufalin-induced apoptosis in A375.S2 human malignant melanoma cells.

Mentions: Results from flow cytometric assay showed that bufalin decreased the levels of ΔΨm (Figure 3(b)). Furthermore, Figure 4(b) indicates that bufalin promoted the levels of cytochrome c, AIF, Endo G, and Bax but inhibited the Bcl-XL expression in A375.S2 cells. Bcl-2 family proteins have been shown to regulate the mitochondria-dependent pathway and death receptor-dependent pathway [43, 44]. Bcl-2 family proteins include the proapoptotic proteins such as Bax, Bak, Bad, and Bcl-Xs and the antiapoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1 [45]. The ratio of Bax/Bcl-2 affects the levels of ΔΨm in cells after exposure to inducer of apoptosis [45], and then mitochondrial release of cytochrome c can be controlled by the ratio of Bax/Bcl-2 proteins and activated by proteolytic cleavage and heterodimerization [46]. Based on those observations, we suggest that bufalin-induced apoptosis in A375.S2 cells was partly through the mitochondria- as well as caspase-dependent and independent pathways. Overall, bufalin induced apoptosis in A375.S2 cells through cross-talk between the extrinsic and the intrinsic pathways. In conclusion, we proposed the possible signaling pathway of bufalin-induced apoptosis in A375.S2 cells that is shown in Figure 5. The possible flow chart indicated that bufalin triggers apoptosis via Fas/FasL, caspase cascade (caspase-3, 8 and 9) or the loss of ΔΨm in mitochondria and then led to AIF and Endo G release that is a novel finding for bufalin-induced apoptosis in A375.S2 cells. These results provided a novel and more detailed molecular mechanism in bufalin-induced apoptosis in A375.S2 cells in vitro.


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)

A schematic diagram showing the proposed signaling pathways of bufalin-induced apoptosis in A375.S2 human malignant melanoma cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: A schematic diagram showing the proposed signaling pathways of bufalin-induced apoptosis in A375.S2 human malignant melanoma cells.
Mentions: Results from flow cytometric assay showed that bufalin decreased the levels of ΔΨm (Figure 3(b)). Furthermore, Figure 4(b) indicates that bufalin promoted the levels of cytochrome c, AIF, Endo G, and Bax but inhibited the Bcl-XL expression in A375.S2 cells. Bcl-2 family proteins have been shown to regulate the mitochondria-dependent pathway and death receptor-dependent pathway [43, 44]. Bcl-2 family proteins include the proapoptotic proteins such as Bax, Bak, Bad, and Bcl-Xs and the antiapoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1 [45]. The ratio of Bax/Bcl-2 affects the levels of ΔΨm in cells after exposure to inducer of apoptosis [45], and then mitochondrial release of cytochrome c can be controlled by the ratio of Bax/Bcl-2 proteins and activated by proteolytic cleavage and heterodimerization [46]. Based on those observations, we suggest that bufalin-induced apoptosis in A375.S2 cells was partly through the mitochondria- as well as caspase-dependent and independent pathways. Overall, bufalin induced apoptosis in A375.S2 cells through cross-talk between the extrinsic and the intrinsic pathways. In conclusion, we proposed the possible signaling pathway of bufalin-induced apoptosis in A375.S2 cells that is shown in Figure 5. The possible flow chart indicated that bufalin triggers apoptosis via Fas/FasL, caspase cascade (caspase-3, 8 and 9) or the loss of ΔΨm in mitochondria and then led to AIF and Endo G release that is a novel finding for bufalin-induced apoptosis in A375.S2 cells. These results provided a novel and more detailed molecular mechanism in bufalin-induced apoptosis in A375.S2 cells in vitro.

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