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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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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-p21WAF1, and anti-GAPDH via 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-f002: 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-p21WAF1, and anti-GAPDH via Western blotting. GAPDH served as the internal control for equal loading. The ratios represent protein alterations compared to the control.

Mentions: Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in the G1 phase of the cycle (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively (Figure 2A,B). In agreement with a previous study [25], there was an increase in p21WAF1 protein levels after crocin treatment in p53-expressing cells, suggesting its role in the observed G1 arrest (Figure 2C). This finding was further confirmed by the continuous decrease of the cell cycle regulators phospho-H3 and Cyclin B1 (Figure 2C). On the other hand, there was a different pattern of cell cycle distribution in the p53-deficient cells. Figure 2A,B show that crocin induced G2 arrest in HCT116 p53−/− after 24 h. Furthermore, 48 h treatment with crocin reinforced the cells to enter M phase and eventually undergo cell death (pre-G1 35.5%) compared to the control (1.9%). The reduction of phospho-H3 would explain why cells stopped cycling after 24 h of crocin treatment (Figure 2C). Crocin treatment after 24 h induced G2 arrest, which was evaluated by reduction in the Cyclin B1 protein level (Figure 2C). At a later time point, there was an increase in phospho-H3 accompanied by regular levels of Cyclin B1 that cells might start recycling after 48 h of crocin treatment, which led to a delayed cell death.


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)

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-p21WAF1, and anti-GAPDH via 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-f002: 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-p21WAF1, and anti-GAPDH via Western blotting. GAPDH served as the internal control for equal loading. The ratios represent protein alterations compared to the control.
Mentions: Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in the G1 phase of the cycle (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively (Figure 2A,B). In agreement with a previous study [25], there was an increase in p21WAF1 protein levels after crocin treatment in p53-expressing cells, suggesting its role in the observed G1 arrest (Figure 2C). This finding was further confirmed by the continuous decrease of the cell cycle regulators phospho-H3 and Cyclin B1 (Figure 2C). On the other hand, there was a different pattern of cell cycle distribution in the p53-deficient cells. Figure 2A,B show that crocin induced G2 arrest in HCT116 p53−/− after 24 h. Furthermore, 48 h treatment with crocin reinforced the cells to enter M phase and eventually undergo cell death (pre-G1 35.5%) compared to the control (1.9%). The reduction of phospho-H3 would explain why cells stopped cycling after 24 h of crocin treatment (Figure 2C). Crocin treatment after 24 h induced G2 arrest, which was evaluated by reduction in the Cyclin B1 protein level (Figure 2C). At a later time point, there was an increase in phospho-H3 accompanied by regular levels of Cyclin B1 that cells might start recycling after 48 h of crocin treatment, which led to a delayed cell death.

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