Apoptosis

, Volume 17, Issue 4, pp 325–334

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

  • Haitao Zhang
  • George G. Chen
  • Zhiyi Zhang
  • Sukying Chun
  • Billy Cheuk Sing Leung
  • Paul B. S. Lai
Original Paper

DOI: 10.1007/s10495-011-0685-y

Cite this article as:
Zhang, H., Chen, G.G., Zhang, Z. et al. Apoptosis (2012) 17: 325. doi:10.1007/s10495-011-0685-y

Abstract

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.

Keywords

SB203580AutophagyHepatocellular carcinomaAMPKDAPKp38 MAPK

Supplementary material

10495_2011_685_MOESM1_ESM.tif (11.4 mb)
Supplemental Fig. 1. Autophagy induced by SB203580. Cells were transfected with GFP-LC3 and treated with 50 μM SB203580 for 24 h. A. Morphologies of cells tested were recorded. B. Intracellular %GFP-LC3-positive cells with GFP-LC3 dots were measured as indicated in Materials and methods. The data represent the mean ± SD, n = 4. aP < 0.01, vs the corresponding control group. (TIFF 11629 kb)
10495_2011_685_MOESM2_ESM.tif (6.6 mb)
Supplemental Fig. 2. The expression of AMPK and DAPK proteins. Cells were treated with 50 μM SB203580 for 24 h and the expression of proteins was determined by Western blot. (TIFF 6752 kb)
10495_2011_685_MOESM3_ESM.tif (1.2 mb)
Supplemental Fig. 3. Effect of SB203580 on cell survival and on cell cycle. A. Cell survival: cells were treated with different concentrations of SB203580 for 24 h. Viable cells were determined by MTT assay. B. Cell cycle: after HepG2 cells were treated with different concentrations of SB203580 for 24 h, they were incubated with propidium iodide and RNase. Cell cycle was analyzed by flow cytometry. The data represent the mean ± SD, n = 4. aP < 0.05, bP < 0.01, cP < 0.001 vs each corresponding control group, respectively. (TIFF 1275 kb)
10495_2011_685_MOESM4_ESM.tif (10.2 mb)
Supplemental Fig. 4. BIRB0796 failed to induce autophagy in HepG2 cells. Cells were transfected with GFP-LC3 and treated with 5 ~ 40 μM BIRB0796 for 24 h, 50 μM SB203580 as positive control. A. Morphology × 200. B. Cells transfected with GFP-LC3 were treated with BIRB0796 and SB203580 for 24 h. C. The expression of proteins was determined by Western blot. D. GFP-LC3-positive cells with GFP-LC3 dots. The data represent mean ± SD, n = 4. bP < 0.01, vs SB203580-treatment group. (TIFF 10494 kb)
10495_2011_685_MOESM5_ESM.tif (14.8 mb)
Supplemental Fig. 5. Over-expression of p38 MAPK unaffected SB203580-induced autophagy. HepG2 cells were transfected with pcDNA3.1-p38 MAPK and treated by 50 μM SB203580. After the treatment, the cell morphology was observed and total protein was isolated for Western blot analysis. A. Morphology × 200. B. Cells transfected with GFP-LC3 and pcDNA3.1-p38 MAPK, then cells were treated SB203580 for another 24 h. C. The expression of proteins was determined by Western blot. D. GFP-LC3-positive cells with GFP-LC3 dots.The data represent mean ± SD, n = 4. aP < 0.01, vs control group; bP < 0.01, vs Control vector-transfected group; cP < 0.01, vs pcDNA3.1-p38 MAPK-transfected group. (TIFF 15198 kb)
10495_2011_685_MOESM6_ESM.tif (23 mb)
Supplemental Fig. 6. The p53 inhibitor PFT-α reduced SB203580-induced autophagy. HepG2 cells were transfected with GFP-LC3 and pre-incubated with 20 μM PFT-α for 2 h, followed by 50 μM SB203580 for 24 h. After the treatment, the cell morphology was observed, intracellular %GFP-LC3-positive cells with GFP-LC3 dots were measured, and total protein was isolated for Western blot analysis. A. Morphology, × 200. B, C. Intracellular %GFP-LC3-positive cells with GFP-LC3 dots. D. The expression of proteins was determined by Western blot. The data represent mean ± SD, n = 4. aP < 0.01, vs control group; bP < 0.05, vs SB203580-treated group. (TIFF 23543 kb)

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Haitao Zhang
    • 1
    • 2
  • George G. Chen
    • 1
  • Zhiyi Zhang
    • 1
  • Sukying Chun
    • 1
  • Billy Cheuk Sing Leung
    • 1
  • Paul B. S. Lai
    • 1
  1. 1.Department of Surgery, Faculty of MedicineThe Chinese University of Hong Kong, Prince of Wales HospitalShatin, New Territories, Hong KongChina
  2. 2.Department of Biochemistry and Molecular BiologyGuangdong Medical CollegeZhanjiangChina