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Ethanol extract of Forsythia suspensa root induces apoptosis of esophageal carcinoma cells via the mitochondrial apoptotic pathway.

Zhao L, Yan X, Shi J, Ren F, Liu L, Sun S, Shan B - Mol Med Rep (2014)

Bottom Line: The results revealed that FSREE, rather than Forsythia suspensa ethanolic extracts from the leaf (FSLEE) and fruit (FSFEE) exhibited marked anti-tumor activity towards human esophageal cancer cells.FSREE induced cancer cell apoptosis and growth arrest by downregulating B‑cell lymphoma (Bcl)‑2, Bcl‑extra large and myeloid cell leukemia 1, while upregulating Bcl‑2‑associated X protein, Bcl‑2 antagonist of cell death and phorbol‑12‑myristate‑13‑acetate‑induced protein 1.Furthermore, the anti-cancer activity of FSREE was associated with a decreased level of phosphorylated Janus kinase/signal transducer and activator of transcription 3 and extracellular‑signal‑regulated kinase signaling activity.

View Article: PubMed Central - PubMed

Affiliation: Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China.

ABSTRACT
Forsythia suspensa root is used in the treatment of fever and jaundice in Traditional Chinese Medicine. In the present study, the anti-tumor activity of the ethanolic extract of Forsythia suspensa root (FSREE) against esophageal carcinoma cells was investigated in vitro and in vivo and its anti-cancer mechanism was examined. The results revealed that FSREE, rather than Forsythia suspensa ethanolic extracts from the leaf (FSLEE) and fruit (FSFEE) exhibited marked anti-tumor activity towards human esophageal cancer cells. FSREE induced cancer cell apoptosis and growth arrest by downregulating B‑cell lymphoma (Bcl)‑2, Bcl‑extra large and myeloid cell leukemia 1, while upregulating Bcl‑2‑associated X protein, Bcl‑2 antagonist of cell death and phorbol‑12‑myristate‑13‑acetate‑induced protein 1. This led to the activation of poly(ADP ribose) polymerase, caspase‑3 and caspase‑9, but not caspase‑8. Furthermore, the anti-cancer activity of FSREE was associated with a decreased level of phosphorylated Janus kinase/signal transducer and activator of transcription 3 and extracellular‑signal‑regulated kinase signaling activity. It was also observed that the levels of cytochrome c were elevated in the cytoplasm, accounting for the loss of mitochondrial membrane potential in the TE‑13 cells upon treatment with FSEER. In addition, FSEER inhibited the growth of esophageal cancer cells in xenograft models and no detectable toxicity was present in the lung or liver tissues. These observations provided further evidence of the anti-tumor effect of FSEER and may be of importance to further examine the potential role of Forsythia suspensa root as a therapeutic agent in esophageal carcinoma therapy.

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Western blot analysis of TE-13 cells treated with FSEER. The TE-13 cells were treated with (A) FSEER at various concentrations for 24 h and (B) 0.5 mg/ml FSEER for 6, 12 and 24 h. Cleaved caspase-3, caspase-9 and PARP were activated and Cyt-c in the cytosolic fraction increased in a dose- and time-dependent manner. *P<0.05, **P<0.01, compared with the control group. FSEER, Forsythia suspensa ethanolic extract of the root; Cyt-c, cytochrome c; PARP, poly(ADP ribose) polymerase.
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f3-mmr-11-02-0871: Western blot analysis of TE-13 cells treated with FSEER. The TE-13 cells were treated with (A) FSEER at various concentrations for 24 h and (B) 0.5 mg/ml FSEER for 6, 12 and 24 h. Cleaved caspase-3, caspase-9 and PARP were activated and Cyt-c in the cytosolic fraction increased in a dose- and time-dependent manner. *P<0.05, **P<0.01, compared with the control group. FSEER, Forsythia suspensa ethanolic extract of the root; Cyt-c, cytochrome c; PARP, poly(ADP ribose) polymerase.

Mentions: Caspase-3 can be activated by a mitochondrial apoptotic pathway involving caspase-9, termed the intrinsic pathway, or by a death receptor pathway involving caspase-8, the extrinsic pathway, contributing to cell apoptosis (12,13). The results of the present study revealed that treatment of TE-13 cells with FSEER for 48 h resulted in cleavage of caspase-3, as evidenced by the appearance of 19-kDa intermediates (Fig. 3A). Furthermore, treatment of the TE-13 cells with FSEER also resulted in significantly increased cleavage of caspase-9 without changes in procaspase-8 levels (Fig. 3B). These results suggested that FRSEE triggered apoptosis through the intrinsic pathway, but not the extrinsic pathway. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including PARP (14). Therefore, PARP has become an essential marker of caspase-3 activity in intrinsic apoptotic pathways (15). As shown in Fig. 3A, the levels of cleaved PARP fragment, which is the active form, were significantly increased following exposure to FSREE for 48 h, further confirming the activity of caspase-3 in the TE-13 cells. In addition, a key step in the intrinsic apoptotic pathway is the damage of mitochondria and the release of Cyt-c to activate apoptotic protease activating factor 1, which in turn activates the caspase cascade (16). Following treatment of TE-13 cells with FSEER, Cyt-c levels increased in the cytoplasmic fraction in a dose- and timedependent manner (Fig. 3). This result indicated that FSEER induced the release of Cyt-c from the mitochondria to the cytoplasm in TE-13 cells and further suggested that the mitochondrial pathway was involved in FSREE-induced apoptosis.


Ethanol extract of Forsythia suspensa root induces apoptosis of esophageal carcinoma cells via the mitochondrial apoptotic pathway.

Zhao L, Yan X, Shi J, Ren F, Liu L, Sun S, Shan B - Mol Med Rep (2014)

Western blot analysis of TE-13 cells treated with FSEER. The TE-13 cells were treated with (A) FSEER at various concentrations for 24 h and (B) 0.5 mg/ml FSEER for 6, 12 and 24 h. Cleaved caspase-3, caspase-9 and PARP were activated and Cyt-c in the cytosolic fraction increased in a dose- and time-dependent manner. *P<0.05, **P<0.01, compared with the control group. FSEER, Forsythia suspensa ethanolic extract of the root; Cyt-c, cytochrome c; PARP, poly(ADP ribose) polymerase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262507&req=5

f3-mmr-11-02-0871: Western blot analysis of TE-13 cells treated with FSEER. The TE-13 cells were treated with (A) FSEER at various concentrations for 24 h and (B) 0.5 mg/ml FSEER for 6, 12 and 24 h. Cleaved caspase-3, caspase-9 and PARP were activated and Cyt-c in the cytosolic fraction increased in a dose- and time-dependent manner. *P<0.05, **P<0.01, compared with the control group. FSEER, Forsythia suspensa ethanolic extract of the root; Cyt-c, cytochrome c; PARP, poly(ADP ribose) polymerase.
Mentions: Caspase-3 can be activated by a mitochondrial apoptotic pathway involving caspase-9, termed the intrinsic pathway, or by a death receptor pathway involving caspase-8, the extrinsic pathway, contributing to cell apoptosis (12,13). The results of the present study revealed that treatment of TE-13 cells with FSEER for 48 h resulted in cleavage of caspase-3, as evidenced by the appearance of 19-kDa intermediates (Fig. 3A). Furthermore, treatment of the TE-13 cells with FSEER also resulted in significantly increased cleavage of caspase-9 without changes in procaspase-8 levels (Fig. 3B). These results suggested that FRSEE triggered apoptosis through the intrinsic pathway, but not the extrinsic pathway. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including PARP (14). Therefore, PARP has become an essential marker of caspase-3 activity in intrinsic apoptotic pathways (15). As shown in Fig. 3A, the levels of cleaved PARP fragment, which is the active form, were significantly increased following exposure to FSREE for 48 h, further confirming the activity of caspase-3 in the TE-13 cells. In addition, a key step in the intrinsic apoptotic pathway is the damage of mitochondria and the release of Cyt-c to activate apoptotic protease activating factor 1, which in turn activates the caspase cascade (16). Following treatment of TE-13 cells with FSEER, Cyt-c levels increased in the cytoplasmic fraction in a dose- and timedependent manner (Fig. 3). This result indicated that FSEER induced the release of Cyt-c from the mitochondria to the cytoplasm in TE-13 cells and further suggested that the mitochondrial pathway was involved in FSREE-induced apoptosis.

Bottom Line: The results revealed that FSREE, rather than Forsythia suspensa ethanolic extracts from the leaf (FSLEE) and fruit (FSFEE) exhibited marked anti-tumor activity towards human esophageal cancer cells.FSREE induced cancer cell apoptosis and growth arrest by downregulating B‑cell lymphoma (Bcl)‑2, Bcl‑extra large and myeloid cell leukemia 1, while upregulating Bcl‑2‑associated X protein, Bcl‑2 antagonist of cell death and phorbol‑12‑myristate‑13‑acetate‑induced protein 1.Furthermore, the anti-cancer activity of FSREE was associated with a decreased level of phosphorylated Janus kinase/signal transducer and activator of transcription 3 and extracellular‑signal‑regulated kinase signaling activity.

View Article: PubMed Central - PubMed

Affiliation: Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China.

ABSTRACT
Forsythia suspensa root is used in the treatment of fever and jaundice in Traditional Chinese Medicine. In the present study, the anti-tumor activity of the ethanolic extract of Forsythia suspensa root (FSREE) against esophageal carcinoma cells was investigated in vitro and in vivo and its anti-cancer mechanism was examined. The results revealed that FSREE, rather than Forsythia suspensa ethanolic extracts from the leaf (FSLEE) and fruit (FSFEE) exhibited marked anti-tumor activity towards human esophageal cancer cells. FSREE induced cancer cell apoptosis and growth arrest by downregulating B‑cell lymphoma (Bcl)‑2, Bcl‑extra large and myeloid cell leukemia 1, while upregulating Bcl‑2‑associated X protein, Bcl‑2 antagonist of cell death and phorbol‑12‑myristate‑13‑acetate‑induced protein 1. This led to the activation of poly(ADP ribose) polymerase, caspase‑3 and caspase‑9, but not caspase‑8. Furthermore, the anti-cancer activity of FSREE was associated with a decreased level of phosphorylated Janus kinase/signal transducer and activator of transcription 3 and extracellular‑signal‑regulated kinase signaling activity. It was also observed that the levels of cytochrome c were elevated in the cytoplasm, accounting for the loss of mitochondrial membrane potential in the TE‑13 cells upon treatment with FSEER. In addition, FSEER inhibited the growth of esophageal cancer cells in xenograft models and no detectable toxicity was present in the lung or liver tissues. These observations provided further evidence of the anti-tumor effect of FSEER and may be of importance to further examine the potential role of Forsythia suspensa root as a therapeutic agent in esophageal carcinoma therapy.

Show MeSH
Related in: MedlinePlus