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

Amin A, Bajbouj K, Koch A, Gandesiri M, Schneider-Stock R - Int J Mol Sci (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Science, United Arab Emirates University, Al-Ain 15551, United Arab Emirates. a.amin@uaeu.ac.ae.

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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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.
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ijms-16-01544-f003: 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.

Mentions: Next we aimed to analyze the possible pro-apoptotic effect of crocin on HCT116 cells by performing Annexin V staining. As shown in Figure 3A,B, there was a pronounced cell death induction in HCT116 p53−/− cells (63%) after 48 h of crocin treatment compared to HCT116 wild-type (11%). Analyzing the caspase 3 protein levels further proved that HCT116 p53−/− cells were more sensitive to crocin treatment. Figure 3C indicates a remarkable cleavage of caspase 3 in HCT116 p53−/− cells, which was associated with a cleavage increase in its downstream target, PARP, compared to the HCT116 wild-type after crocin treatment. Moreover, there was no indication of DNA damage, measured by the amount of γH2AX in the HCT116 wild-type cells after crocin treatment (Figure 3D). As expected, in p53-deficient cells, the observed cell death was accompanied by an increased DNA damage detected by a notable increase in γH2AX, which was significantly intensified after 6 h of crocin treatment in a time-dependent manner (Figure 3D).


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

Amin A, Bajbouj K, Koch A, Gandesiri M, Schneider-Stock R - Int J Mol Sci (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307319&req=5

ijms-16-01544-f003: 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.
Mentions: Next we aimed to analyze the possible pro-apoptotic effect of crocin on HCT116 cells by performing Annexin V staining. As shown in Figure 3A,B, there was a pronounced cell death induction in HCT116 p53−/− cells (63%) after 48 h of crocin treatment compared to HCT116 wild-type (11%). Analyzing the caspase 3 protein levels further proved that HCT116 p53−/− cells were more sensitive to crocin treatment. Figure 3C indicates a remarkable cleavage of caspase 3 in HCT116 p53−/− cells, which was associated with a cleavage increase in its downstream target, PARP, compared to the HCT116 wild-type after crocin treatment. Moreover, there was no indication of DNA damage, measured by the amount of γH2AX in the HCT116 wild-type cells after crocin treatment (Figure 3D). As expected, in p53-deficient cells, the observed cell death was accompanied by an increased DNA damage detected by a notable increase in γH2AX, which was significantly intensified after 6 h of crocin treatment in a time-dependent manner (Figure 3D).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Science, United Arab Emirates University, Al-Ain 15551, United Arab Emirates. a.amin@uaeu.ac.ae.

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.

Show MeSH
Related in: MedlinePlus