J Breast Cancer.  2015 Jun;18(2):112-118. 10.4048/jbc.2015.18.2.112.

Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells

• Zhang X
• Luo W
• Zhao W
• Lu J
• Chen X

• KMID: 2374525
• DOI: http://doi.org/10.4048/jbc.2015.18.2.112

Abstract

PURPOSE
Isocryptotanshinone (ICTS) is a natural bioactive product that is isolated from the roots of the widely used medical herb Salvia miltiorrhiza. However, few reports exist on the mechanisms underlying the therapeutic effects of ICTS. Here, we report that ICTS has anticancer activity and describe the mechanism underlying this effect.
METHODS
The antiproliferative effect of ICTS was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and clonogenic assays. The effect of ICTS on the cell cycle was measured using flow cytometry. Apoptosis was determined by Hoechst 33342 staining, DNA fragmentation assays, and Western blotting for apoptotic proteins. Finally, the effect of ICTS on mitogen-activated protein kinases (MAPKs) was determined by Western blotting.
RESULTS
ICTS significantly inhibited proliferation of MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 human liver cancer cells, and A549 human lung cancer cells in vitro. Among the tested cell lines, MCF-7 cells showed the highest sensitivity to ICTS. ICTS significantly inhibited colony formation by MCF-7 cells. Furthermore, exposure of MCF-7 cells to ICTS induced cell cycle arrest at the G1 phase and decreased mitochondrial membrane potential. Hoechst 33342 staining and Western blot analysis for apoptotic proteins suggested that ICTS induced apoptosis in MCF-7 cells. In addition, ICTS activated MAPK signaling in MCF-7 cells by inducing time- and concentration-dependent phosphorylation of JNK, ERK, and p38 MAPK.
CONCLUSION
Our results suggest that ICTS inhibited MCF-7 cell proliferation by inducing apoptosis and activating MAPK signaling pathways.

Keyword

Apoptosis; Breast neoplasms; Isocryptotanshinone; Mitogen-activated protein kinases

MeSH Terms

Apoptosis*
Blotting, Western
Breast Neoplasms*
Cell Cycle
Cell Cycle Checkpoints
Cell Line
DNA Fragmentation
Flow Cytometry
G1 Phase
Hep G2 Cells
Humans
Liver Neoplasms
Lung Neoplasms
MCF-7 Cells*
Membrane Potential, Mitochondrial
Mitogen-Activated Protein Kinases
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Salvia miltiorrhiza
Mitogen-Activated Protein Kinases
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 The chemical structure and cytotoxicity of isocryptotanshinone (ICTS). (A) The chemical structure of ICTS. (B) MCF-7, MDA-MB-231, HepG2, and A549 cells were treated with indicated concentrations (0-40 µM) of ICTS for 24 hours. Cell viabilities were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. (C) After treatment with ICTS (0-20 µM) for 24 hours, morphological changes of MCF-7 cells were photographed. (D) MCF-7 cells were treated with indicated concentrations of ICTS for 24 hours. At a concentration of 20 µM, ICTS almost completely inhibited colony formation by MCF-7 cells.

• 

  Figure 2 Effect of isocryptotanshinone (ICTS) on cell cycle distribution of MCF-7 cells. (A) Cells were treated with ICTS (0-10 µM) for 24 hours, and cell cycle distribution was analyzed using flow cytometer. (B) The percentages of each G1, S, and G2/M phase for different treatment. *p<0.05 compared with control group.

• 

  Figure 3 Isocryptotanshinone (ICTS) induced apoptosis in MCF-7 cells. (A) Cells were treated with indicated concentrations of ICTS for 24 hours, and the nuclei were stained by Hoechst 33342. Arrows indicated the condensed nuclei in cells. (B) DNA fragmentation assay was performed in MCF-7 cells after treatment with indicated concentrations of ICTS for 24 hours. (C, D) MCF-7 cells were treated with ICTS (0-20 µM) for 24 hours, and the expressions of Bcl-2, Bcl-XL, BAK, and BAX, as well as poly-ADP-ribose polymerase (PARP), caspase-3, and caspase-9 were detected by Western blot. GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

• 

  Figure 4 Effect of isocryptotanshinone (ICTS) on mitochondrial membrane potential (MMP). MCF-7 cells were treated with 20 µM ICTS for 0, 2, 4, 8, and 20 hours, the MMP was determined by flow cytometer using JC-1 staining. Cells treated with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 20 minutes were used as the positive control. *p<0.05; †p<0.01 compared with 0 hour treatment group.

• 

  Figure 5 Effect of isocryptotanshinone (ICTS) on the mitogen activated protein kinases signaling. (A) MCF-7 cells were treated with indicated concentrations of ICTS for 24 hours, and the expressions of total and phosphorylated JNK, ERK, p38 were detected by Western blot. Glycer-aldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. (B) Cells were treated with ICTS for 0, 1, 3, 6, 12, and 24 hours, respectively, the expressions of p-JNK, p-ERK, and p-p38 were detected by Western blot. GAPDH was used as internal control.

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