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

Zhang X, Luo W, Zhao W, Lu J, Chen X - J Breast Cancer (2015)

Bottom Line: Apoptosis was determined by Hoechst 33342 staining, DNA fragmentation assays, and Western blotting for apoptotic proteins.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.Hoechst 33342 staining and Western blot analysis for apoptotic proteins suggested that ICTS induced apoptosis in MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: To determine whether ICTS induced apoptosis in MCF-7 cells, nuclear fluorescent staining and DNA fragmentation assays were conducted. As shown in Figure 3A, after treatment with ICTS, morphological characteristics indicative of apoptotic cells, including nuclear condensation and fragmentation, were observed in Hoechst 33342-stained MCF-7 cells. Although the DNA fragmentation assay did not show clear "DNA ladders," diffuse fragmentation was observed (Figure 3B). Furthermore, expression levels of apoptosis-related proteins were determined. As shown in Figure 3C, after ICTS treatment, the abundance of antiapoptotic proteins Bcl-2 and Bcl-XL decreased, while that of proapoptotic proteins BAX and BAK increased. In addition, a remarkable increase in the abundance of cleaved PARP, cleaved caspase-3, and caspase-9 was also detected after ICTS treatment (Figure 3D). These results confirmed that ICTS induced apoptosis in MCF-7 cells.


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

Zhang X, Luo W, Zhao W, Lu J, Chen X - J Breast Cancer (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490259&req=5

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.
Mentions: To determine whether ICTS induced apoptosis in MCF-7 cells, nuclear fluorescent staining and DNA fragmentation assays were conducted. As shown in Figure 3A, after treatment with ICTS, morphological characteristics indicative of apoptotic cells, including nuclear condensation and fragmentation, were observed in Hoechst 33342-stained MCF-7 cells. Although the DNA fragmentation assay did not show clear "DNA ladders," diffuse fragmentation was observed (Figure 3B). Furthermore, expression levels of apoptosis-related proteins were determined. As shown in Figure 3C, after ICTS treatment, the abundance of antiapoptotic proteins Bcl-2 and Bcl-XL decreased, while that of proapoptotic proteins BAX and BAK increased. In addition, a remarkable increase in the abundance of cleaved PARP, cleaved caspase-3, and caspase-9 was also detected after ICTS treatment (Figure 3D). These results confirmed that ICTS induced apoptosis in MCF-7 cells.

Bottom Line: Apoptosis was determined by Hoechst 33342 staining, DNA fragmentation assays, and Western blotting for apoptotic proteins.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.Hoechst 33342 staining and Western blot analysis for apoptotic proteins suggested that ICTS induced apoptosis in MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.

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.

No MeSH data available.


Related in: MedlinePlus