Effector mechanism of magnolol-induced apoptosis in human lung squamous carcinoma CH27 cells

Abstract

1 Magnolol, an active component isolated from the root and stem bark of Magnolia officinalis, has been reported to exhibit antitumour effects, but little is known about its molecular mechanisms of action. 2 Magnolol inhibited proliferation of human lung squamous carcinoma CH27 cells at low concentrations (10-40 microM), and induced apoptosis at high concentrations (80-100 microM). 3 Treatment with 80 microM magnolol significantly increased the expression of Bad and Bcl-X(S) proteins, whereas it decreased the expression of Bcl-X(L). Overexpression of Bcl-2 protected CH27 cells against magnolol-triggered apoptosis. 4 Magnolol treatment resulted in accumulation of cytosolic cytochrome c and activation of caspase-9 and downstream caspases (caspase-3 and -6). Pretreatment with z-VAD-fmk markedly inhibited magnolol-induced cell death, but did not prevent cytosolic cytochrome c accumulation. 5 Magnolol induced a modest and persistent JNK activation and ERK inactivation in CH27 cells without evident changes in the protein levels. The responsiveness of JNK and ERK to magnolol suggests the involvement of these kinases in the initiation of the apoptosis process. 6 These results indicate that regulation of the Bcl-2 family, accumulation of cytosolic cytochrome c, and activation of caspase-9 and caspase-3 may be the effector mechanisms of magnolol-induced apoptosis.

Figure 1

Magnolol-mediated cytotoxicity in CH27, H460, H1299 and WI-38 cells. (A) Dose-dependent response. Cells were treated with various concentrations of magnolol for 24 h. After incubation, the cells were harvested and cell viability was counted by Trypan blue dye exclusion method as described in Methods. (B) Time-dependent response. Cells were treated with control vehicle or 80 μM magnolol for the indicated times. After treatment, cell viability was determined as described above. Data are presented as mean±s.d. of 12 replicates from four separate experiments.

Figure 2

Induction of apoptosis by magnolol. (A) Morphological changes. CH27, H460, H1299 and WI-38 cells were treated with control vehicle (0.1% DMSO) or 80 μM magnolol for 24 h. Phase contrast micrographs were shown (magnification ×200; scale bar, 20 μm). (B) Condensed nuclei. CH27 cells were treated with 80 μM magnolol for 24 h, fixed and stained with DAPI, or using TUNEL assay, then examined under a fluorescence microscope. All photographs were taken at magnification ×200; scale bar, 20 μm. (C) DNA fragmentation analysis. Adherent and floating CH27 cells were collected 24 h after 80 μM magnolol treatment. Total DNA was isolated and separated by 2% agarose gel electrophoresis. M, size marker (100 base-pair DNA ladder); lane 1, control CH27 cells; lane 2, 80 μM magnolol-treated CH27 cells.

Figure 3

Expression of Bcl-2 family proteins in magnolol-treated CH27 cells. After treatment without or with 80 μM magnolol for indicated time periods, total cellular proteins were extracted and assayed by Western blot, using the antibodies against specific Bcl-2 family proteins. β-Actin was used as an internal loading control.

Figure 4

Overexpression of Bcl-2 protein prevented magnolol-induced apoptosis. CH27 cells were infected with Adv-Bcl-2 or Adv-vector for 24 h, then treated with 80 μM magnolol for another 24 h. Total cellular extracts were examined using Bcl-2 specific antibody by Western blot, cell viability was measured by Trypan blue exclusion method.

Figure 5

Effect of magnolol on caspase activity and cytochrome c. (A) Caspase activity analysis. Extracts from untreated or magnolol-treated CH27 cells were assayed for caspase activity using fluorogenic peptide substrates. (B) Proteolytic cleavage of caspase-3 and PARP were analysed by Western blot analysis. β-Actin was used as an internal loading control. (C) Inhibition of magnolol-induced apoptotic cell death by z-VAD-fmk. CH27 cells were treated with cell permeable broad-spectrum caspase inhibitor (z-VAD-fmk) 2 h prior to 80 μM magnolol treatment. Cell viability was estimated. ^***P<0.001, compared with the 80 μM magnolol-treated group. (D) Effect of magnolol on cytosolic cytochrome c. Cytosolic fractions were isolated and analysed by Western blot for cytochrome c specific antibody. (E) z-VAD-fmk did not affect magnolol-induced cytosolic cytochrome c accumulation. CH27 cells were pretreated with 100 μM z-VAD-fmk for 2 h, then treated with vehicle or with 80 μM magnolol for 8 or 16 h. After treatment, cytosolic extracts were analysed by Western blot for cytochrome c.

Figure 6

Regulation of MAPK molecules by magnolol. CH27 cells were treated with 80 μM magnolol for the indicated times. Cell extracts were analysed by Western blot, using the indicated antibodies. β-Actin was used as an internal loading control.

Figure 7

Effect of magnolol on c-Jun and the JNK activity. CH27 cells were treated with 80 μM magnolol for the indicated times. (A) Phosphorylated c-Jun was analysed by Western blot using phospho-c-Jun specific antibody. (B) JNK activity was measured in kinase reaction buffer using GST-c-Jun as substrate.