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

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.

Keywords: AGS human gastric adenocarcinoma cells; Rheum undulatum L; anti-proliferative activity; apoptosis; cell death; intrinsic apoptotic pathway.

Fig. 1. Induction of apoptosis by MERL treatment in AGS cells. The cells were treated with the indicated concentrations of MERL for 24 hours. The cell viability was measured by using a MTT assay. Bars represent the mean ± S.D. ^*P < 0.05. ^†P < 0.01. MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cells; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; S.D., standard deviation.

Fig. 2. MERL increases the activity of sub G1 phase cells in AGS cells. To quantify the degree of apoptosis induced by MERL, we evaluated the cells by using flow cytometry to determine the sub G1 DNA content, which represents the cells undergoing apoptotic DNA degradation. Bars represent the mean ± SD. ^*P < 0.05. ^†P < 0.01. MERL, methanol extract of Rheum undulatum L.; AGS, adenocarcinoma gastric cells; S.D., standard deviation.

Fig. 3. 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.

Fig. 4. Effects of MERL on the MMP values and the levels of tBid, Bcl-2 and Bax proteins in AGS cells. (A) Cells were treated with the indicated concentration of MERL for 24 hours. Cells were collected and incubated with JC-1 (10 μM) for 20 minutes at 37˚C in the dark. The cells were the washed once with PBS and analyzed by a DNA flow cytometer. (B) The cell lysates obtained from cells grown under the same conditions as (A) were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Actin was used as an internal control. Bars represent the mean ± S.D. ^*P < 0.01. MERL, methanol extract of Rheum undulatum L.; MMP, mitochondrial membrane potential; tBid, truncated Bid; Bcl-2, B-cell lymphoma 2; Bax, Bcl2 Antagonist X; AGS, adenocarcinoma gastric cell lines; JC-1, 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; S.D., standard deviation.

Fig. 5. 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.