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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

Effects of a specific inhibitor of MAPK on MERL induced cell death in AGS cells. (A) Cells were pretreated with the indicated MAPK inhibitors (SB203580 (20 μM), SP600125 (20 μM) and PD98059 (50 μM)) for 1 hour and then treated with MERL (350 μg/mL) for 24 hours. The cell’s viability was measured by using the MTT assay. Bars represent the mean ± SD. *P < 0.01. MAPK, mitogen activated protein kinase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; S.D., standard deviation; n.s., not significant vs. MERL treated cells.
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Figure 005: Effects of a specific inhibitor of MAPK on MERL induced cell death in AGS cells. (A) Cells were pretreated with the indicated MAPK inhibitors (SB203580 (20 μM), SP600125 (20 μM) and PD98059 (50 μM)) for 1 hour and then treated with MERL (350 μg/mL) for 24 hours. The cell’s viability was measured by using the MTT assay. Bars represent the mean ± SD. *P < 0.01. MAPK, mitogen activated protein kinase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; S.D., standard deviation; n.s., not significant vs. MERL treated cells.

Mentions: The MAPK signaling pathway functions as a critical regulator of cell survival and proliferation [20]. The MAPKs, including p38 MAPK, ERKs and JNKs also play fundamental roles in survival, proliferation, and apoptosis [21]. To determine whether these signaling pathways played a role in the MERL induced apoptotic response, we pretreated the cells with a specific inhibitor of MAPK and then measured the cell’s viability by using the MTT assay. As shown in (Fig. 5), pretreatment with SB203580 (a specific inhibitor of p38 MAPK), SP600125 (a potent inhibitor of JNK), or PD98059 (a potent inhibitor of ERK) did not have an effect on the response to MERL treatment. Taken together, these results suggest that MERL induced cell death is associated with the intrinsic apoptotic pathway 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)

Effects of a specific inhibitor of MAPK on MERL induced cell death in AGS cells. (A) Cells were pretreated with the indicated MAPK inhibitors (SB203580 (20 μM), SP600125 (20 μM) and PD98059 (50 μM)) for 1 hour and then treated with MERL (350 μg/mL) for 24 hours. The cell’s viability was measured by using the MTT assay. Bars represent the mean ± SD. *P < 0.01. MAPK, mitogen activated protein kinase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; S.D., standard deviation; n.s., not significant vs. MERL treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 005: Effects of a specific inhibitor of MAPK on MERL induced cell death in AGS cells. (A) Cells were pretreated with the indicated MAPK inhibitors (SB203580 (20 μM), SP600125 (20 μM) and PD98059 (50 μM)) for 1 hour and then treated with MERL (350 μg/mL) for 24 hours. The cell’s viability was measured by using the MTT assay. Bars represent the mean ± SD. *P < 0.01. MAPK, mitogen activated protein kinase; MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cell lines; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; S.D., standard deviation; n.s., not significant vs. MERL treated cells.
Mentions: The MAPK signaling pathway functions as a critical regulator of cell survival and proliferation [20]. The MAPKs, including p38 MAPK, ERKs and JNKs also play fundamental roles in survival, proliferation, and apoptosis [21]. To determine whether these signaling pathways played a role in the MERL induced apoptotic response, we pretreated the cells with a specific inhibitor of MAPK and then measured the cell’s viability by using the MTT assay. As shown in (Fig. 5), pretreatment with SB203580 (a specific inhibitor of p38 MAPK), SP600125 (a potent inhibitor of JNK), or PD98059 (a potent inhibitor of ERK) did not have an effect on the response to MERL treatment. Taken together, these results suggest that MERL induced cell death is associated with the intrinsic apoptotic pathway 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