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Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers.

Li Z, Dong Z, Myer D, Yip-Schneider M, Liu J, Cui P, Schmidt CM, Zhang JT - BMC Cancer (2010)

Bottom Line: In the present study, we tested this hypothesis.A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays.We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA. jianzhan@iupui.edu.

ABSTRACT

Background: Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis.

Methods: Western blot analysis was used to determine 14-3-3σ protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays.

Results: We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3σ in a pancreatic cancer cell line caused resistance to γ-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest.

Conclusion: The increased level of 14-3-3σ protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3σ may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients.

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14-3-3σ over-expression increased drug-induced G2/M arrest and resistance to drug-induced apoptosis. A, Cell cycle distribution analysis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 5 Gy γ-irradiation (IR), 10 nM mitoxantrone, or control without treatment followed by analysis of cell cycle distribution using FACS. B, Effect of 14-3-3σ radiation-induced apoptosis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with (filled bars) or without (white bars) γ-irradiation followed by analysis of cells at sub-G1 phase using FACS. C and D, Effect of 14-3-3σ on radiation- and drug-induced PARP cleavage. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 20 Gy γ-irradiation (C) or 10 μM mitoxantrone (D) for the times indicated followed by analysis of PARP cleavage using Western blot analysis. The graph in panel D shows the rate of production of the 85-kDa PARP fragments product (as a ratio of the 85-kDa fragment to the full-length 115-kDa protein).
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Figure 6: 14-3-3σ over-expression increased drug-induced G2/M arrest and resistance to drug-induced apoptosis. A, Cell cycle distribution analysis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 5 Gy γ-irradiation (IR), 10 nM mitoxantrone, or control without treatment followed by analysis of cell cycle distribution using FACS. B, Effect of 14-3-3σ radiation-induced apoptosis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with (filled bars) or without (white bars) γ-irradiation followed by analysis of cells at sub-G1 phase using FACS. C and D, Effect of 14-3-3σ on radiation- and drug-induced PARP cleavage. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 20 Gy γ-irradiation (C) or 10 μM mitoxantrone (D) for the times indicated followed by analysis of PARP cleavage using Western blot analysis. The graph in panel D shows the rate of production of the 85-kDa PARP fragments product (as a ratio of the 85-kDa fragment to the full-length 115-kDa protein).

Mentions: Previously, we observed that over-expression of 14-3-3σ increases G2/M arrest upon DNA damage in prostate cancer cells as a mechanism of survival, by allowing more time to repair DNA damage [10]. To determine if the over-expression of 14-3-3σ also causes similar G2/M arrest upon DNA damage in pancreatic cancer cells, we treated the stable 14-3-3σ-over-expressing and vec-transfected control cells with γ-irradiation or mitoxantrone followed by FACS analysis of cell cycle distribution. As shown in Figure 6A, the stable 14-3-3σ-over-expressing cells clearly have a much higher proportion of cells arrested at G2/M than the vector-transfected control cells following γ-irradiation or mitoxantrone treatment. Thus, in pancreatic cancer cells 14-3-3σ likely provides a protection mechanism by arresting cells at G2/M phase, thereby providing cells an opportunity to repair DNA damage and survive DNA-damaging treatments.


Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers.

Li Z, Dong Z, Myer D, Yip-Schneider M, Liu J, Cui P, Schmidt CM, Zhang JT - BMC Cancer (2010)

14-3-3σ over-expression increased drug-induced G2/M arrest and resistance to drug-induced apoptosis. A, Cell cycle distribution analysis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 5 Gy γ-irradiation (IR), 10 nM mitoxantrone, or control without treatment followed by analysis of cell cycle distribution using FACS. B, Effect of 14-3-3σ radiation-induced apoptosis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with (filled bars) or without (white bars) γ-irradiation followed by analysis of cells at sub-G1 phase using FACS. C and D, Effect of 14-3-3σ on radiation- and drug-induced PARP cleavage. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 20 Gy γ-irradiation (C) or 10 μM mitoxantrone (D) for the times indicated followed by analysis of PARP cleavage using Western blot analysis. The graph in panel D shows the rate of production of the 85-kDa PARP fragments product (as a ratio of the 85-kDa fragment to the full-length 115-kDa protein).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2991307&req=5

Figure 6: 14-3-3σ over-expression increased drug-induced G2/M arrest and resistance to drug-induced apoptosis. A, Cell cycle distribution analysis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 5 Gy γ-irradiation (IR), 10 nM mitoxantrone, or control without treatment followed by analysis of cell cycle distribution using FACS. B, Effect of 14-3-3σ radiation-induced apoptosis. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with (filled bars) or without (white bars) γ-irradiation followed by analysis of cells at sub-G1 phase using FACS. C and D, Effect of 14-3-3σ on radiation- and drug-induced PARP cleavage. Vector- and 14-3-3σ-transfected stable MiaPaCa-2 cells were treated with 20 Gy γ-irradiation (C) or 10 μM mitoxantrone (D) for the times indicated followed by analysis of PARP cleavage using Western blot analysis. The graph in panel D shows the rate of production of the 85-kDa PARP fragments product (as a ratio of the 85-kDa fragment to the full-length 115-kDa protein).
Mentions: Previously, we observed that over-expression of 14-3-3σ increases G2/M arrest upon DNA damage in prostate cancer cells as a mechanism of survival, by allowing more time to repair DNA damage [10]. To determine if the over-expression of 14-3-3σ also causes similar G2/M arrest upon DNA damage in pancreatic cancer cells, we treated the stable 14-3-3σ-over-expressing and vec-transfected control cells with γ-irradiation or mitoxantrone followed by FACS analysis of cell cycle distribution. As shown in Figure 6A, the stable 14-3-3σ-over-expressing cells clearly have a much higher proportion of cells arrested at G2/M than the vector-transfected control cells following γ-irradiation or mitoxantrone treatment. Thus, in pancreatic cancer cells 14-3-3σ likely provides a protection mechanism by arresting cells at G2/M phase, thereby providing cells an opportunity to repair DNA damage and survive DNA-damaging treatments.

Bottom Line: In the present study, we tested this hypothesis.A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays.We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA. jianzhan@iupui.edu.

ABSTRACT

Background: Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis.

Methods: Western blot analysis was used to determine 14-3-3σ protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays.

Results: We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3σ in a pancreatic cancer cell line caused resistance to γ-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest.

Conclusion: The increased level of 14-3-3σ protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3σ may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients.

Show MeSH
Related in: MedlinePlus