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

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.

Figure 1

Effect of increasing concentrations of crocin on the growth of HCT116 wild-type (wt) and HCT116 p53−/− cells for 24 and 48 h. (A,B) Viability test assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showing the HCT116 wild-type and HCT116 p53−/− cells untreated (ctrl) or treated with different concentrations of crocin (Cro; 0.5 to 15 mM) for 24 and 48 h. The MTT data shown are performed in quadruplicates; and (C) Cell numbers (dead and alive) and (D) cell death percentages were measured using the trypan blue staining after crocin treatment for 24 and 48 h (* p < 0.05).

Figure 1

Effect of increasing concentrations of crocin on the growth of HCT116 wild-type (wt) and HCT116 p53−/− cells for 24 and 48 h. (A,B) Viability test assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showing the HCT116 wild-type and HCT116 p53−/− cells untreated (ctrl) or treated with different concentrations of crocin (Cro; 0.5 to 15 mM) for 24 and 48 h. The MTT data shown are performed in quadruplicates; and (C) Cell numbers (dead and alive) and (D) cell death percentages were measured using the trypan blue staining after crocin treatment for 24 and 48 h (* p < 0.05).

Figure 2

Cell cycle arrest after crocin administration. (A) Two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with 10 mM crocin for 24 and 48 h, were harvested and DNA was stained with PI for flow cytometric analysis of DNA content with FACScan flow cytometry; (B) Quantitative analysis of percentage gated cells at sub-G₁, G₁, S, and G₂ phases in the HCT116 wild-type and HCT116 p53−/− cells treated with 10 mM crocin for 24 and 48 h; and (C) Lysates prepared from two cell types, HCT116 wild-type and HCT116 p53−/−, untreated (Ctrl) or treated with 10 mM crocin for 6, 24, and 48 h were analyzed by anti-H3, anti-phospho Histone 3 (pH3), anti-Cyclin B1, anti-p21^WAF1, and anti-GAPDH via Western blotting. GAPDH served as the internal control for equal loading. The ratios represent protein alterations compared to the control.

Figure 3

Apoptosis induction after crocin treatment. (A) Annexin-PI measurements of untreated cells (Ctrl) and two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, treated with 10 mM crocin for 6, 24, and 48 h. The profile represents Annexin-V-FITC staining on the x-axis and PI on the y-axis; (B) Quantitative analysis of percentage gated for viable, necrotic, early apoptotic, and late apoptotic HCT116 wild-type (wt) and HCT116 p53−/− cells treated with 10 mM crocin for 6, 24, and 48 h; (C) 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-caspase3, anti-PARP, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading; and (D) 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-γH2AX, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading. The ratios represent protein alterations compared to the control.

Figure 3

Apoptosis induction after crocin treatment. (A) Annexin-PI measurements of untreated cells (Ctrl) and two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, treated with 10 mM crocin for 6, 24, and 48 h. The profile represents Annexin-V-FITC staining on the x-axis and PI on the y-axis; (B) Quantitative analysis of percentage gated for viable, necrotic, early apoptotic, and late apoptotic HCT116 wild-type (wt) and HCT116 p53−/− cells treated with 10 mM crocin for 6, 24, and 48 h; (C) 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-caspase3, anti-PARP, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading; and (D) 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-γH2AX, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading. The ratios represent protein alterations compared to the control.

Figure 4

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.

Figure 5

Crocin-induced autophagy in a p53-dependent manner. (A) Lysates of HCT116 wildtype and HCT116 p53−/−, untreated (Ctrl) or treated with Bafilomycin A1 (10 nM) for 1 h and/or further treated with 10 mM crocin for 48 h, were analyzed by anti-cyclin B1, anti-phospho Histone 3 (pH3) and anti-GAPDH via Western blotting. GAPDH served as internal control for equal loading; (B) Quantitative analysis of percentage gated viable, necrotic, early apoptotic, and late necrotic cells in the HCT116 wild-type (wt) and HCT116 p53−/− cells treated with Bafilomycin A1 (Baf, 10 nM) for 1 h and/or further treated with 10 mM crocin (Cro) for 48 h, measured by Annexin-PI staining; (C) Lysates prepared from two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with 10 mM crocin for 24 h and/or further treated with bafilomycin (10 nM) for 48 h, were analyzed by anti-caspase3, anti-PARP, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading; and (D) Lysates prepared from two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with Bafilomycin A1 (10 nM) for 1 h and/or further treated with 10 mM crocin for 48 h, were analyzed by anti-γH2AX and anti-GAPDH in Western blotting. The ratios represent protein alterations compared to the control.

Figure 5

Crocin-induced autophagy in a p53-dependent manner. (A) Lysates of HCT116 wildtype and HCT116 p53−/−, untreated (Ctrl) or treated with Bafilomycin A1 (10 nM) for 1 h and/or further treated with 10 mM crocin for 48 h, were analyzed by anti-cyclin B1, anti-phospho Histone 3 (pH3) and anti-GAPDH via Western blotting. GAPDH served as internal control for equal loading; (B) Quantitative analysis of percentage gated viable, necrotic, early apoptotic, and late necrotic cells in the HCT116 wild-type (wt) and HCT116 p53−/− cells treated with Bafilomycin A1 (Baf, 10 nM) for 1 h and/or further treated with 10 mM crocin (Cro) for 48 h, measured by Annexin-PI staining; (C) Lysates prepared from two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with 10 mM crocin for 24 h and/or further treated with bafilomycin (10 nM) for 48 h, were analyzed by anti-caspase3, anti-PARP, and anti-GAPDH Western blotting. GAPDH served as the internal control for equal loading; and (D) Lysates prepared from two cell types, HCT116 wild-type (wt) and HCT116 p53−/−, untreated (Ctrl) or treated with Bafilomycin A1 (10 nM) for 1 h and/or further treated with 10 mM crocin for 48 h, were analyzed by anti-γH2AX and anti-GAPDH in Western blotting. The ratios represent protein alterations compared to the control.