J Vet Sci.  2009 Mar;10(1):23-28. 10.4142/jvs.2009.10.1.23.

Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells

• Cho HS
• Chang SH
• Chung YS
• Shin JY
• Park SJ
• Lee ES
• Hwang SK
• Kwon JT
• Tehrani AM
• Woo M
• Noh MS
• Hanifah H
• Jin H
• Xu CX
• Cho MH

• KMID: 1089342
• DOI: http://doi.org/10.4142/jvs.2009.10.1.23

Abstract

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to have anti-tumor activity in various malignant neoplasms. However, the precise mechanism by which TET inhibits tumor cell growth remains to be elucidated. The present studies were performed to characterize the potential effects of TET on phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase (ERK) pathways since these signaling pathways are known to be responsible for cell growth and survival. TET suppressed cell proliferation and induced apoptosis in A549 human lung carcinoma cells. TET treatment resulted in a down-regulation of Akt and ERK phosphorylation in both time-/concentration-dependent manners. The inhibition of ERK using PD98059 synergistically enhanced the TET-induced apoptosis of A549 cells whereas the inhibition of Akt using LY294002 had a less significant effect. Taken together, our results suggest that TET: i) selectively inhibits the proliferation of lung cancer cells by blocking Akt activation and ii) increases apoptosis by inhibiting ERK. The treatment of lung cancers with TET may enhance the efficacy of chemotherapy and radiotherapy and increase the apoptotic potential of lung cancer cells.

Keyword

A549 cells; Akt; apoptosis; Erk; tetrandrine

MeSH Terms

Antineoplastic Agents, Phytogenic/*pharmacology
Apoptosis/drug effects
Benzylisoquinolines/*pharmacology
Carcinoma/*drug therapy
Cell Line, Tumor
Dose-Response Relationship, Drug
Extracellular Signal-Regulated MAP Kinases/*antagonists & inhibitors
Humans
Lung Neoplasms/*drug therapy

Figure

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  Fig. 1 The effect of tetrandrine on the proliferation of A549 cells. The viability of A549 cells was measured using the MTT assay. The cells were incubated with increasing concentrations of tetrandrine for (A) 24 h or (B) 48 h. Data are presented as mean ± SE of 3 independent experiments. *p < 0.05, **p < 0.01.

• 

  Fig. 2 Flowcytometric detection of apoptosis of A549 cells treated with tetrandrine (TET). Cells were incubated with 30 µM of TET for 12 h and 24 h. (A) Control, (B) TET 12 h (C) TET 24 h, (D) Percentage of apoptotic cells from the time-dependent study. *p < 0.05, **p < 0.01.

• 

  Fig. 3 The effect of tetrandrine (TET) on the levels of pro- and anti-apoptotic proteins in A549 cells. Cells were treated with (A) various concentrations (0, 10, 20 and 30 µM) of TET for 24 h or (B) 30 µM of TET for indicated times (0, 2, 4, 8, 12 and 24 h).

• 

  Fig. 4 The effect of tetrandrine (TET) on Akt activation in A549 cells. The cells were treated with (A) various concentrations (0, 10, 20 and 30 µM) of TET for 24 h or (B) 30 µM of TET for indicated times (0, 2, 4, 8, 12 and 24 h).

• 

  Fig. 5 The effect of tetrandrine (TET) on ERK activation in A549 cells. The cells were treated with (A) various concentrations (0, 10, 20 and 30 µM) of TET for 24 h or (B) 30 µM TET for indicated times (0, 2, 4, 8, 12 and 24 h).

• 

  Fig. 6 Flowcytometric detection of apoptosis in A549 cells. Cells were treated with tetrandrine (TET) (30 µM) for 24 h in the absence or presence of LY294002 (20 µM) or PD98059 (50 µM). (A) Control, (B) TET (30 µM), (C) TET (30 µM) + LY294002 (20 µM), (D) TET (30 µM) + PD98059 (50 µM), (E) Summary of percentage of apoptotic cells in the inhibitor study. Data are presented as mean ± SE of 3 independent experiments. **p < 0.01.

• 

  Fig. 7 The effects of a PI3K/Akt inhibitor and an MEK/ERK inhibitor on tetrandrine (TET)-treated A549 cells. The cells were treated with TET (30 µM) for 24 h in the absence or presence of LY294002 (20 µM) or PD98059 (50 µM). Next, lysates were prepared and Western blot analysis was performed in order to determine protein expression levels.

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