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Defective autophagosome formation in p53-null colorectal cancer reinforces crocin-induced apoptosis

Amin A1, Bajbouj K2, Koch A3, Gandesiri M4, Schneider-Stock R5. Int J Mol Sci. 2015 ;16(1):1544-61.

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1Department of Biology, College of Science, United Arab Emirates University, Al-Ain 15551, United Arab Emirates. [email protected]

2Department of Biology, College of Science, United Arab Emirates University, Al-Ain 15551, United Arab Emirates. [email protected]

3Experimental Tumor Pathology, Institute of Pathology, University of Erlangen, Erlangen 91054, Germany. [email protected]

4Experimental Tumor Pathology, Institute of Pathology, University of Erlangen, Erlangen 91054, Germany. [email protected]

5Experimental Tumor Pathology, Institute of Pathology, University of Erlangen, Erlangen 91054, Germany. [email protected]

 

Abstract

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53(-/-) cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53(-/-) after 24 h. Crocin induced inefficient autophagy in HCT116 p53(-/-) cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation.  Autophagosome formation was not detected in HCT116 p53(-/-) after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53(-/-) cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death.

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Figure Legend:

Crocin-induced autophagy in HCT116 cells. (A) Lysates prepared from two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with 10 mM crocin for 6, 24, and 48 h, were analyzed by anti-LC3, anti-Beclin 1, anti-Atg7, anti-p62, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading; (B) Fluorescence staining of LC3 and DAPI in two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, control and treated with 10 mM crocin for 24 h; and (C) Lysates prepared from two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with Bafilomycin A1 (Baf, 10 nM) for 1 h and/or further treated with 10 mM crocin (Cro) for 48 h were analyzed by anti-LC3, anti-p62, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading. The ratios represent protein alterations compared to the control.

Defective Autophagosome Formation in p53-Null Colorectal Cancer Reinforces Crocin-Induced Apoptosis. Global Medical Discovery