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Association of a Methanol Extract of Rheum undulatum L. Mediated Cell Death in AGS Cells with an Intrinsic Apoptotic Pathway.

Hong NR, Park HS, Ahn TS, Jung MH, Kim BJ - J Pharmacopuncture (2015)

Bottom Line: Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control.However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Longevity and Biofunctional Medicine, Healthy Aging Korean Medical Research Center, School of Korean Medicine, Pusan National University, Busan, Korea.

ABSTRACT

Objectives: Rheum undulatum L. has traditionally been used for the treatment of many diseases in Asia. However, its anti-proliferative activity in cancer has still not been studied. In the present study, we investigated the anti-cancer effects of methanol extract of Rheum undulatum L. (MERL) on human adenocarcinoma gastric cell lines (AGS).

Methods: To investigate the anti-cancer effect of MERL on AGS cells, we treated the AGS cells with varying con¬centrations of MERL and performed 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Cell cycle analyses, measurements of the mitochondrial membrane potential (MMP), caspase activity assays and Western blots were conducted to determine whether AGS cell death occurred by apoptosis.

Results: Treatment with MERL significantly inhibited growth of AGS cells in a concentration dependent manner. MERL treatment in AGS cells leaded to increased accumulation of apoptotic sub G1 phase cells in a concentration dependent manner. In control cultures, 5.38% of the cells were in the sub G1 phase. In MERL treated cells, however, this percentage was significantly increased (9.95% at 70 μg/mL, 15.94% at 140 μg/mL, 26.56% at 210 μg/mL and 38.08% at 280 μg/mL). MERL treatment induced the decreased expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the expression of the active form of caspase-3 was increased. A subsequent Western blot analysis revealed increased cleaved levels of poly (ADP-ribose) polymerase (PARP) protein. Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control. MERL treatment increased the levels of the pro-apoptotic truncated Bid (tBid) and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, whose is the stabilization of mitochondria. However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.

Conclusion: These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

No MeSH data available.


Related in: MedlinePlus

Activation of caspases and degradation of the PARP protein due to MERL treatment of AGS cells. (A) AGS cells were treated with the indicated concentrations of MERL for 24 hours. Actin was used as an internal control. (B) After a 24 hours incubation with the indicated concentrations of MERL, the cells were lysed and aliquots were assayed for the in-vitro caspase-3 and -9 activities with DEVD-pNA and LEHD-pNA as substrates, respectively. Bars represent the mean ± SD. *P < 0.05. †P < 0.01. PARP, polymerase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; DEVD, Asp-Glu-Val-Asp; p-NA, p-nitroaniline; LEHD, Leu-Glu-His-Asp; S.D., standard deviation.
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Figure 003: Activation of caspases and degradation of the PARP protein due to MERL treatment of AGS cells. (A) AGS cells were treated with the indicated concentrations of MERL for 24 hours. Actin was used as an internal control. (B) After a 24 hours incubation with the indicated concentrations of MERL, the cells were lysed and aliquots were assayed for the in-vitro caspase-3 and -9 activities with DEVD-pNA and LEHD-pNA as substrates, respectively. Bars represent the mean ± SD. *P < 0.05. †P < 0.01. PARP, polymerase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; DEVD, Asp-Glu-Val-Asp; p-NA, p-nitroaniline; LEHD, Leu-Glu-His-Asp; S.D., standard deviation.

Mentions: Caspases are known to be important mediators of apoptosis in both the intrinsic and the extrinsic pathways. This executionary phase induces activation of cytoplasmic endonuclease, which degrades nuclear material, activation of proteases, which degrades cytoskeletal proteins and nuclei, and cleavage of various substrates including polymerase (PARP), which serves as an apoptosis marker [15]. As shown in Fig. 3a, MERL treatment induced a decrease in the expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the active form of caspase-3 was increased. Subsequent Western blot analyses revealed increased cleaved levels of the PARP protein. We next attempted to quantify the proteolytic activation of caspases due to the MERL treatment. As shown in Fig. 3b, treatment with MERL increased the activities of caspase-3 and -9 compared with the levels for the controls. These results indicate that MERL treatment induces apoptosis via activation of caspases in AGS cells.


Association of a Methanol Extract of Rheum undulatum L. Mediated Cell Death in AGS Cells with an Intrinsic Apoptotic Pathway.

Hong NR, Park HS, Ahn TS, Jung MH, Kim BJ - J Pharmacopuncture (2015)

Activation of caspases and degradation of the PARP protein due to MERL treatment of AGS cells. (A) AGS cells were treated with the indicated concentrations of MERL for 24 hours. Actin was used as an internal control. (B) After a 24 hours incubation with the indicated concentrations of MERL, the cells were lysed and aliquots were assayed for the in-vitro caspase-3 and -9 activities with DEVD-pNA and LEHD-pNA as substrates, respectively. Bars represent the mean ± SD. *P < 0.05. †P < 0.01. PARP, polymerase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; DEVD, Asp-Glu-Val-Asp; p-NA, p-nitroaniline; LEHD, Leu-Glu-His-Asp; S.D., standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481396&req=5

Figure 003: Activation of caspases and degradation of the PARP protein due to MERL treatment of AGS cells. (A) AGS cells were treated with the indicated concentrations of MERL for 24 hours. Actin was used as an internal control. (B) After a 24 hours incubation with the indicated concentrations of MERL, the cells were lysed and aliquots were assayed for the in-vitro caspase-3 and -9 activities with DEVD-pNA and LEHD-pNA as substrates, respectively. Bars represent the mean ± SD. *P < 0.05. †P < 0.01. PARP, polymerase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; DEVD, Asp-Glu-Val-Asp; p-NA, p-nitroaniline; LEHD, Leu-Glu-His-Asp; S.D., standard deviation.
Mentions: Caspases are known to be important mediators of apoptosis in both the intrinsic and the extrinsic pathways. This executionary phase induces activation of cytoplasmic endonuclease, which degrades nuclear material, activation of proteases, which degrades cytoskeletal proteins and nuclei, and cleavage of various substrates including polymerase (PARP), which serves as an apoptosis marker [15]. As shown in Fig. 3a, MERL treatment induced a decrease in the expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the active form of caspase-3 was increased. Subsequent Western blot analyses revealed increased cleaved levels of the PARP protein. We next attempted to quantify the proteolytic activation of caspases due to the MERL treatment. As shown in Fig. 3b, treatment with MERL increased the activities of caspase-3 and -9 compared with the levels for the controls. These results indicate that MERL treatment induces apoptosis via activation of caspases in AGS cells.

Bottom Line: Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control.However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Longevity and Biofunctional Medicine, Healthy Aging Korean Medical Research Center, School of Korean Medicine, Pusan National University, Busan, Korea.

ABSTRACT

Objectives: Rheum undulatum L. has traditionally been used for the treatment of many diseases in Asia. However, its anti-proliferative activity in cancer has still not been studied. In the present study, we investigated the anti-cancer effects of methanol extract of Rheum undulatum L. (MERL) on human adenocarcinoma gastric cell lines (AGS).

Methods: To investigate the anti-cancer effect of MERL on AGS cells, we treated the AGS cells with varying con¬centrations of MERL and performed 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Cell cycle analyses, measurements of the mitochondrial membrane potential (MMP), caspase activity assays and Western blots were conducted to determine whether AGS cell death occurred by apoptosis.

Results: Treatment with MERL significantly inhibited growth of AGS cells in a concentration dependent manner. MERL treatment in AGS cells leaded to increased accumulation of apoptotic sub G1 phase cells in a concentration dependent manner. In control cultures, 5.38% of the cells were in the sub G1 phase. In MERL treated cells, however, this percentage was significantly increased (9.95% at 70 μg/mL, 15.94% at 140 μg/mL, 26.56% at 210 μg/mL and 38.08% at 280 μg/mL). MERL treatment induced the decreased expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the expression of the active form of caspase-3 was increased. A subsequent Western blot analysis revealed increased cleaved levels of poly (ADP-ribose) polymerase (PARP) protein. Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control. MERL treatment increased the levels of the pro-apoptotic truncated Bid (tBid) and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, whose is the stabilization of mitochondria. However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.

Conclusion: These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

No MeSH data available.


Related in: MedlinePlus