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Selenocysteine derivative overcomes TRAIL resistance in melanoma cells: evidence for ROS-dependent synergism and signaling crosstalk.

Cao W, Li X, Zheng S, Zheng W, Wong YS, Chen T - Oncotarget (2014)

Bottom Line: Moreover, silencing of p53 down-regulated the expression levels of p53-inducible genes, and effectively blocked the cell apoptosis.Suppression of PI3K significantly increased the apoptotic cell death.In contrast, antioxidants effectively reversed the cell apoptosis through regulation of Akt and p53 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Jinan University, Guangzhou, China.

ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as one of the most promising targeted drug for new cancer therapeutics, is limited in clinical application by the evolution of resistance in many cancer cell lines, especially in malignant melanoma. Thus, it is urgently needed to identify chemosensitizers to enhance the apoptotic inducing efficacy of TRAIL and overcome resistance of malignant melanoma cells. Herein, we reported that 3,3'-diselenodipropionic acid (DSeA), a Selenocysteine derivative, could synergistically enhance the growth inhibitory effect of TRAIL on A375 melanoma cells though induction of ROS-dependent apoptosis with involvement of PTEN-mediated Akt inactivation and DNA damage-mediated p53 phosphorylation, which subsequently activated mitochondrial and death receptor apoptotic pathways. Moreover, silencing of p53 down-regulated the expression levels of p53-inducible genes, and effectively blocked the cell apoptosis. Suppression of PI3K significantly increased the apoptotic cell death. In contrast, antioxidants effectively reversed the cell apoptosis through regulation of Akt and p53 signaling pathways. Taken together, the combination of DSeA and TRAIL could be a novel strategy to overcome TRAIL resistance in malignant melanoma, and DSeA may be candidates for further evaluation as a chemosensitizer in clinical trails.

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Related in: MedlinePlus

Co-treatment activated DNA damage-mediated p53-dependent apoptotic pathways in A375 cells(A) Western blot analysis of expression levels of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53 in A375 cells exposed to 20 μM DSeA for 24 h and/or 40 ng/TRAIL for another 24 h. (B) The time course of regulation of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53, Bcl-xl and Bax in A375 cells induced by DSeA. Cells were treated with 20 μM DSeA and harvested at various times. (C) Co-treatment-induced DNA damage in A375 cells. Cells after treatment were immediately analyzed by Comet assay as described in Methods. The length of tail reflects the degree of DNA damage in cells. (D, E) Protective effects of p53 siRNA on co-treatment-induced cell growth inhibition and caspases activation in A375 cells. Cells were pretreated with 50 nmol p53 siRNA for 24 h, and then treated in combination with DSeA and TRAIL. Cell viability was examined by MTT assay. Bars with different characters are statistically different at p < 0.05 level. Caspase activity was measured using synthetic fluorogenic substrate as described in Methods. Each value represents the mean ± SD of three independent experiments, *, p < 0.05; **, p < 0.01 versus the control. (F) Western blot analysis of the inhibitory effects of p53 siRNA on co-treatment-induced cell apoptosis, expression and activation of p53 and caspases.
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Figure 4: Co-treatment activated DNA damage-mediated p53-dependent apoptotic pathways in A375 cells(A) Western blot analysis of expression levels of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53 in A375 cells exposed to 20 μM DSeA for 24 h and/or 40 ng/TRAIL for another 24 h. (B) The time course of regulation of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53, Bcl-xl and Bax in A375 cells induced by DSeA. Cells were treated with 20 μM DSeA and harvested at various times. (C) Co-treatment-induced DNA damage in A375 cells. Cells after treatment were immediately analyzed by Comet assay as described in Methods. The length of tail reflects the degree of DNA damage in cells. (D, E) Protective effects of p53 siRNA on co-treatment-induced cell growth inhibition and caspases activation in A375 cells. Cells were pretreated with 50 nmol p53 siRNA for 24 h, and then treated in combination with DSeA and TRAIL. Cell viability was examined by MTT assay. Bars with different characters are statistically different at p < 0.05 level. Caspase activity was measured using synthetic fluorogenic substrate as described in Methods. Each value represents the mean ± SD of three independent experiments, *, p < 0.05; **, p < 0.01 versus the control. (F) Western blot analysis of the inhibitory effects of p53 siRNA on co-treatment-induced cell apoptosis, expression and activation of p53 and caspases.

Mentions: Bcl-2 family members have been described as key regulators of mitochondrial permeability [25]. Therefore, we examined the effects of DSeA and/or TRAIL on the expression levels of pro-survival and pro-apoptotic Bcl-2 family proteins in A375 cells. As shown in Fig. 3C and Fig. 3D, Western blot analysis revealed that co-treatment significantly suppressed the expression of pro-survival Bcl-2 family proteins, such as Bcl-2, Mcl-1, and Bcl-xl, and up-regulated the expression of pro-apoptosis Bcl-2 family proteins, such as Bax, Bad, Bim and PUMA. Moreover, dephosphorylation of Bad at serine 112 was also observed in response to the combined treatment. The time course analysis showed that the alternation of the expression levels of Bcl-2 family proteins could be detected after treated with DSeA for 4 h (Fig. 4B). These results indicate that the combined treatment causes the depletion of Δψm by regulating the expression of Bcl-2 family proteins.


Selenocysteine derivative overcomes TRAIL resistance in melanoma cells: evidence for ROS-dependent synergism and signaling crosstalk.

Cao W, Li X, Zheng S, Zheng W, Wong YS, Chen T - Oncotarget (2014)

Co-treatment activated DNA damage-mediated p53-dependent apoptotic pathways in A375 cells(A) Western blot analysis of expression levels of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53 in A375 cells exposed to 20 μM DSeA for 24 h and/or 40 ng/TRAIL for another 24 h. (B) The time course of regulation of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53, Bcl-xl and Bax in A375 cells induced by DSeA. Cells were treated with 20 μM DSeA and harvested at various times. (C) Co-treatment-induced DNA damage in A375 cells. Cells after treatment were immediately analyzed by Comet assay as described in Methods. The length of tail reflects the degree of DNA damage in cells. (D, E) Protective effects of p53 siRNA on co-treatment-induced cell growth inhibition and caspases activation in A375 cells. Cells were pretreated with 50 nmol p53 siRNA for 24 h, and then treated in combination with DSeA and TRAIL. Cell viability was examined by MTT assay. Bars with different characters are statistically different at p < 0.05 level. Caspase activity was measured using synthetic fluorogenic substrate as described in Methods. Each value represents the mean ± SD of three independent experiments, *, p < 0.05; **, p < 0.01 versus the control. (F) Western blot analysis of the inhibitory effects of p53 siRNA on co-treatment-induced cell apoptosis, expression and activation of p53 and caspases.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Co-treatment activated DNA damage-mediated p53-dependent apoptotic pathways in A375 cells(A) Western blot analysis of expression levels of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53 in A375 cells exposed to 20 μM DSeA for 24 h and/or 40 ng/TRAIL for another 24 h. (B) The time course of regulation of phosphorylated ATR, p53, Histone H2A.X, MDM2 and Total p53, Bcl-xl and Bax in A375 cells induced by DSeA. Cells were treated with 20 μM DSeA and harvested at various times. (C) Co-treatment-induced DNA damage in A375 cells. Cells after treatment were immediately analyzed by Comet assay as described in Methods. The length of tail reflects the degree of DNA damage in cells. (D, E) Protective effects of p53 siRNA on co-treatment-induced cell growth inhibition and caspases activation in A375 cells. Cells were pretreated with 50 nmol p53 siRNA for 24 h, and then treated in combination with DSeA and TRAIL. Cell viability was examined by MTT assay. Bars with different characters are statistically different at p < 0.05 level. Caspase activity was measured using synthetic fluorogenic substrate as described in Methods. Each value represents the mean ± SD of three independent experiments, *, p < 0.05; **, p < 0.01 versus the control. (F) Western blot analysis of the inhibitory effects of p53 siRNA on co-treatment-induced cell apoptosis, expression and activation of p53 and caspases.
Mentions: Bcl-2 family members have been described as key regulators of mitochondrial permeability [25]. Therefore, we examined the effects of DSeA and/or TRAIL on the expression levels of pro-survival and pro-apoptotic Bcl-2 family proteins in A375 cells. As shown in Fig. 3C and Fig. 3D, Western blot analysis revealed that co-treatment significantly suppressed the expression of pro-survival Bcl-2 family proteins, such as Bcl-2, Mcl-1, and Bcl-xl, and up-regulated the expression of pro-apoptosis Bcl-2 family proteins, such as Bax, Bad, Bim and PUMA. Moreover, dephosphorylation of Bad at serine 112 was also observed in response to the combined treatment. The time course analysis showed that the alternation of the expression levels of Bcl-2 family proteins could be detected after treated with DSeA for 4 h (Fig. 4B). These results indicate that the combined treatment causes the depletion of Δψm by regulating the expression of Bcl-2 family proteins.

Bottom Line: Moreover, silencing of p53 down-regulated the expression levels of p53-inducible genes, and effectively blocked the cell apoptosis.Suppression of PI3K significantly increased the apoptotic cell death.In contrast, antioxidants effectively reversed the cell apoptosis through regulation of Akt and p53 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Jinan University, Guangzhou, China.

ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as one of the most promising targeted drug for new cancer therapeutics, is limited in clinical application by the evolution of resistance in many cancer cell lines, especially in malignant melanoma. Thus, it is urgently needed to identify chemosensitizers to enhance the apoptotic inducing efficacy of TRAIL and overcome resistance of malignant melanoma cells. Herein, we reported that 3,3'-diselenodipropionic acid (DSeA), a Selenocysteine derivative, could synergistically enhance the growth inhibitory effect of TRAIL on A375 melanoma cells though induction of ROS-dependent apoptosis with involvement of PTEN-mediated Akt inactivation and DNA damage-mediated p53 phosphorylation, which subsequently activated mitochondrial and death receptor apoptotic pathways. Moreover, silencing of p53 down-regulated the expression levels of p53-inducible genes, and effectively blocked the cell apoptosis. Suppression of PI3K significantly increased the apoptotic cell death. In contrast, antioxidants effectively reversed the cell apoptosis through regulation of Akt and p53 signaling pathways. Taken together, the combination of DSeA and TRAIL could be a novel strategy to overcome TRAIL resistance in malignant melanoma, and DSeA may be candidates for further evaluation as a chemosensitizer in clinical trails.

Show MeSH
Related in: MedlinePlus