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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 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.
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ijms-16-01544-f005: 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.

Mentions: It is widely known that autophagy could play a role in either anti-apoptotic or pro-apoptotic pathways. Annexin V staining was analyzed to explore whether autophagy might be involved in the observed different patterns of induced apoptosis after Baf treatment and with or without crocin exposure. Figure 5A shows that Baf pretreatment enhanced the apoptosis induction in HCT116 wild-type cells, whereas there were only minor changes in the massive induced cell death in HCT116 p53−/− after crocin exposure. Moreover, inhibiting autophagosome-lysosome complex formation in HCT116 wild-type cells led to a pronounced cleavage of caspase 3 and PARP, which was associated with an increase in γH2AX protein levels after crocin exposure, suggesting an autophagy-driven pro-survival role of crocin when p53 is functional (Figure 5B). Conversely, Baf-exposed HCT116 p53−/− cells did not show a notable enhancement of apoptosis induction through an increased cleavage of caspase 3 or PARP after crocin treatment (Figure 5B). The increase in the DNA damage sensor γH2AX may suggest a pro-survival role of autophagy (Figure 5C). Thus, crocin induced apoptosis or autophagy, depending on the p53 status of the cells.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4307319&req=5

ijms-16-01544-f005: 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.
Mentions: It is widely known that autophagy could play a role in either anti-apoptotic or pro-apoptotic pathways. Annexin V staining was analyzed to explore whether autophagy might be involved in the observed different patterns of induced apoptosis after Baf treatment and with or without crocin exposure. Figure 5A shows that Baf pretreatment enhanced the apoptosis induction in HCT116 wild-type cells, whereas there were only minor changes in the massive induced cell death in HCT116 p53−/− after crocin exposure. Moreover, inhibiting autophagosome-lysosome complex formation in HCT116 wild-type cells led to a pronounced cleavage of caspase 3 and PARP, which was associated with an increase in γH2AX protein levels after crocin exposure, suggesting an autophagy-driven pro-survival role of crocin when p53 is functional (Figure 5B). Conversely, Baf-exposed HCT116 p53−/− cells did not show a notable enhancement of apoptosis induction through an increased cleavage of caspase 3 or PARP after crocin treatment (Figure 5B). The increase in the DNA damage sensor γH2AX may suggest a pro-survival role of autophagy (Figure 5C). Thus, crocin induced apoptosis or autophagy, depending on the p53 status of the cells.

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