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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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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).
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ijms-16-01544-f001: 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).

Mentions: The effect of crocin on HCT116 cell lines’ cell viability was examined by MTT analysis. Crocin reduced proliferation in a time- and dose-dependent manner. Figure 1A,B show a significant, but different cell proliferation inhibition (p < 0.05) of both HCT116 wild-type and HCT116 p53−/− cell lines at a concentration of 10 mM of crocin after 24 h, of 40% and 65%, respectively. However, both cell lines showed the same pattern of reduced cell proliferation (65%) after 48 h of crocin treatment. To justify this result, trypan blue staining was conducted for both cell lines treated with 10 mM crocin for 24 and 48 h. Indeed, crocin induced more cell death among the surviving 30% (Figure 1B) HCT116 p53−/− cells compared to HCT116 wild-type cells after 24 and 48 h (Figure 1C,D). This is in agreement with a previous study showing that crocin-mediated cell viability inhibition was affected by p53 status [24].


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01544-f001: 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).
Mentions: The effect of crocin on HCT116 cell lines’ cell viability was examined by MTT analysis. Crocin reduced proliferation in a time- and dose-dependent manner. Figure 1A,B show a significant, but different cell proliferation inhibition (p < 0.05) of both HCT116 wild-type and HCT116 p53−/− cell lines at a concentration of 10 mM of crocin after 24 h, of 40% and 65%, respectively. However, both cell lines showed the same pattern of reduced cell proliferation (65%) after 48 h of crocin treatment. To justify this result, trypan blue staining was conducted for both cell lines treated with 10 mM crocin for 24 and 48 h. Indeed, crocin induced more cell death among the surviving 30% (Figure 1B) HCT116 p53−/− cells compared to HCT116 wild-type cells after 24 and 48 h (Figure 1C,D). This is in agreement with a previous study showing that crocin-mediated cell viability inhibition was affected by p53 status [24].

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