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Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

Tor YS, Yazan LS, Foo JB, Wibowo A, Ismail N, Cheah YK, Abdullah R, Ismail M, Ismail IS, Yeap SK - PLoS ONE (2015)

Bottom Line: EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05).The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent.The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.

ABSTRACT
Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.

No MeSH data available.


Related in: MedlinePlus

Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk a indicates statistically significantly different from the untreated control (P<0.05).
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pone.0127441.g006: Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk a indicates statistically significantly different from the untreated control (P<0.05).

Mentions: Western blot analysis was employed to determine the effect of EADs on the expression of proteins involved in apoptotic pathway. Fig 6A depicts that after treatment with EADs, the expression level of p21 and p53 was upregulated, but caspase-8 was downregulated at 24 and 48 hours. Following treatment with EADs, the expression level of Bax and Bcl-2 was upregulated at 24 hours but downregulated at 48 hours (P<0.05). Fig 6B shows that the ratio of Bax to Bcl-2 in EADs-treated MCF-7 cells was higher compared to the control (P<0.05). The expression of total-Akt was downregulated. Meanwhile, the expression of phosphorylated AKT increased at 24 hours, but declined at 48 hours. The expression level of total-JNK-1 and phosphorylated JNK-1 increased significantly compared to the control at 48 hours (P<0.05) (Fig 6C). On the other hand, significant downregulation in the level of phosphorylated ERK was only observed at 48 hours (P<0.05).


Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

Tor YS, Yazan LS, Foo JB, Wibowo A, Ismail N, Cheah YK, Abdullah R, Ismail M, Ismail IS, Yeap SK - PLoS ONE (2015)

Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk a indicates statistically significantly different from the untreated control (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4457850&req=5

pone.0127441.g006: Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk a indicates statistically significantly different from the untreated control (P<0.05).
Mentions: Western blot analysis was employed to determine the effect of EADs on the expression of proteins involved in apoptotic pathway. Fig 6A depicts that after treatment with EADs, the expression level of p21 and p53 was upregulated, but caspase-8 was downregulated at 24 and 48 hours. Following treatment with EADs, the expression level of Bax and Bcl-2 was upregulated at 24 hours but downregulated at 48 hours (P<0.05). Fig 6B shows that the ratio of Bax to Bcl-2 in EADs-treated MCF-7 cells was higher compared to the control (P<0.05). The expression of total-Akt was downregulated. Meanwhile, the expression of phosphorylated AKT increased at 24 hours, but declined at 48 hours. The expression level of total-JNK-1 and phosphorylated JNK-1 increased significantly compared to the control at 48 hours (P<0.05) (Fig 6C). On the other hand, significant downregulation in the level of phosphorylated ERK was only observed at 48 hours (P<0.05).

Bottom Line: EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05).The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent.The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.

ABSTRACT
Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.

No MeSH data available.


Related in: MedlinePlus