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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: 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.

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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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.
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ijms-16-01544-f004: 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.

Mentions: We have previously shown that p53 played a role in autophagy induction after saffron treatment [8]. Since crocin is a major active ingredient of saffron [26], we aimed to examine whether the p53 status could mediate autophagy induction in HCT116 cells, by analyzing autophagy-regulatory proteins LC3, Beclin 1, Atg7, and p62. As shown in Figure 4A, there was a conversion of LC3-I to LC3-II in HCT116 wild-type cells after crocin treatment, which was associated with unchanged protein levels of Beclin 1 and Atg7. Moreover, we did not detect any dramatic degradation of p62 (Figure 4A). Although autophagosome formation was confirmed by measuring LC3-II staining by immunofluorescence staining (Figure 4B), we suggest that there was only a slight autophagy induction after crocin treatment. In contrast to HCT116 wild-type cells, crocin induced a remarkable formation of LC3-II in HCT116 p53−/− cells after 24 and 48 h, which was combined with a decrease in the protein levels of Beclin 1and Atg7 later after 48 h, whereas there was no clear p62 degradation at any time point (Figure 4A). Surprisingly, although there was more LC3-II conversion, we did not observe any punctuation of LC3-II staining in HCT116 p53−/− after crocin treatment (Figure 4B), predicting a non-functional autophagosome formation.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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ijms-16-01544-f004: 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.
Mentions: We have previously shown that p53 played a role in autophagy induction after saffron treatment [8]. Since crocin is a major active ingredient of saffron [26], we aimed to examine whether the p53 status could mediate autophagy induction in HCT116 cells, by analyzing autophagy-regulatory proteins LC3, Beclin 1, Atg7, and p62. As shown in Figure 4A, there was a conversion of LC3-I to LC3-II in HCT116 wild-type cells after crocin treatment, which was associated with unchanged protein levels of Beclin 1 and Atg7. Moreover, we did not detect any dramatic degradation of p62 (Figure 4A). Although autophagosome formation was confirmed by measuring LC3-II staining by immunofluorescence staining (Figure 4B), we suggest that there was only a slight autophagy induction after crocin treatment. In contrast to HCT116 wild-type cells, crocin induced a remarkable formation of LC3-II in HCT116 p53−/− cells after 24 and 48 h, which was combined with a decrease in the protein levels of Beclin 1and Atg7 later after 48 h, whereas there was no clear p62 degradation at any time point (Figure 4A). Surprisingly, although there was more LC3-II conversion, we did not observe any punctuation of LC3-II staining in HCT116 p53−/− after crocin treatment (Figure 4B), predicting a non-functional autophagosome formation.

Bottom Line: 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.

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