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1.
Figure 10

Figure 10. Mechanisms by which imatinib abrogates intrinsic doxorubicin resistance.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

Imatinib prevents activation of STAT3, which promotes doxorubicin-mediated NF-κB phosphorylation and nuclear translocation and NF-κB-mediated inhibition of expression of anti-apoptotic proteins such as cIAP1/XIAP. Imatinib-mediated inhibition of STAT3 also prevents activation of a HSP27/p38/Akt pro-survival pathway.

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
2.
Figure 3

Figure 3. Imatinib reverses doxorubicin resistance by inhibiting cell survival.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

Caspase-3/7 activity (A,B,E), Annexin V staining (C), or PARP cleavage (D,F) were assessed in lysates from attached and detached treated cells (40 h unless otherwise indicated). Mean±SEM of 3 experiments (A,B,E) or representative of 3 independent experiments (C,D,F). Some error bars (A,B,E) are too small to visualize. *p<0.05, **p≤0.01, ***p<0.001 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
3.
Figure 5

Figure 5. Resistance to doxorubicin occurs via ABCB1-dependent and ABCB1-independent mechanisms.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A, B) ABCB1 was inhibited with verapamil (A) or silenced with siRNA (B), cells were treated with imatinib (10 µM) and/or doxorubicin (0.5 µM) for 72 h, and cell viability assessed. Mean±SEM from 3 independent experiments. (A) Dox+imatinib, CI = 0.66±0.19; Dox+verapamil, CI = 0.14±0.04; Dox+imatinib+verapamil, CI = 0.47±0.5. Representative Western blot (right). Some error bars are too small to visualize.

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
4.
Figure 8

Figure 8. Imatinib promotes p65 nuclear translocation and inhibits NF-κB target expression in a STAT3-dependent manner.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A) 435s/M14 cells, transfected with STAT3 siRNA, were drug treated (8 h), and nuclear fractions analyzed by Western blot. Graph is Mean±SEM from 3 independent experiments. (B) 435s/M14 cells stably expressing pcDNA or STAT3C were drug treated (8 h), and nuclear fractions analyzed by Western blot. Graph is Mean±SEM of 3 independent experiments. (C) Lysates from drug-treated pcDNA and STAT3C-expressing cells (40 h), were analyzed by Western blot. Graphs are Mean±SEM from 3 independent experiments. For all subfigures, some error bars are too small to visualize. *p<0.05, **p≤0.01 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
5.
Figure 6

Figure 6. Imatinib promotes apoptosis in response to doxorubicin by inhibiting STAT3-dependent survival pathways.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A) 435s/M14 cells, stably transfected with pcDNA or STAT3C, were treated with imatinib (40 h), and caspase 3/7 activity assessed in lysate from attached/detached cells. Graph is Mean±SEM from 3 independent experiments. Representative Western blot (above). (B) Parental (435s/M14) and acquired doxorubicin-resistant (435s/M14-DR) cells were drug-treated (40 h), and lysates from attached/detached cells were blotted with antibodies. Graphs are Mean±SEM from 3 independent experiments. (C) Western blots of lysates from 435s/M14 cells expressing pcDNA or STAT3C, treated with imatinib/doxorubicin (40 h). Graphs are Mean±SEM from 3 independent experiments. (D) Lysates from drug-treated (40 h), attached/detatched 435s/M14 cells transfected with STAT3 siRNA were blotted. Graphs are Mean±SEM from 3 independent experiments. For all subfigures, some error bars are too small to visualize. *p<0.05, **p≤0.01, ***p<0.001 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
6.
Figure 7

Figure 7. Imatinib abrogates doxorubicin resistance, in part, by increasing p65 nuclear translocation, and inhibiting expression of NF-κB targets.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A) Nuclear lysates from doxorubin (0.5 µM-top; 1 µM-bottom)/imatinib (10 µM)-treated 435s/M14 cells (8 h) were blotted. Graphs are Mean±SEM from 3 independent experiments. (B) Parental (435s/M14) and doxorubicin-resistant (435s/M14-DR) cells, stably expressing a 3X-NF-κB-luciferase reporter, were drug treated (8 h), and luciferase activity assessed. Graphs are Mean±SEM from 3 independent experiments. (C, D) Lysates from treated cells were analyzed by Western blot (40 h treatment). Representative blots are shown. Mean±SEM from 3 independent experiments is shown in Figure S6D,E. (E) 435s/M14 cells transfected with p65 siRNA were drug-treated (8 h), and PARP and caspase-3 cleavage assessed. Graphs are Mean±SEM from 3 independent experiments. *p<0.05, **p≤0.01 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
7.
Figure 2

Figure 2. Imatinib abrogates doxorubicin resistance, in part, by potentiating doxorubicin-mediated G2/M arrest.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A–C) Proliferation was assessed in treated cells (72 h) by tritiated thymidine assay. Representative dose response curves (A, B, left) and Mean±SEM for 3 independent experiments (A–C, right). Some error bars are too small to visualize. (C) CI values: Dox(0.015 µM)+imatinib(2 µM) = 0.9±0.14; Dox(0.015 µM)+imatinib(5 µM) = 0.67±0.07; Dox(0.015 µM)+imatinib(10 µM) = 0.5±0.06; Dox(0.03 µM)+imatinib(2 µM) = 0.8±0.11; Dox(0.03 µM)+imatinib(5 µM) = 0.5±0.13; Dox(0.03 µM)+imatinib(10 µM) = 0.36±0.18. (D, E) Asynchronous drug-treated cells (72 h) were analyzed by FACS. Experiments were performed ≥3 times, and representative cell cycle distributions are shown. Mean±SEM for 3 independent experiments is shown in Figure S4E. **p≤0.01, ***p<0.001 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
8.
Figure 9

Figure 9. Imatinib abrogates doxorubicin resistance, in part, by preventing activation of a STAT3/HSP27/p38/Akt survival pathway.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A, B) 435s/M14 cells were treated with imatinib (10 µM) (A) or transfected with STAT3 siRNA (B), treated with doxorubicin (p38, Akt, 0.5 µM; HSP27, 1 µM) for 40 h, and lysate from attached/detached cells blotted with antibodies. Experiments were performed 3 times; representative blots are shown. Mean±SEM for 3 experiments is shown in Figure S7A. (C) 435s/M14 cells expressing pcDNA or STAT3C were drug treated (40 h), and lysate from attached/detached cells were blotted with antibodies. Graphs are Mean±SEM from 3 independent experiments (bottom). (D) 435s/M14 cells expressing vector or constitutively active PI3K (E545K) were drug-treated (8 h) and PARP cleavage assessed. Graph is Mean±SEM from 3 independent experiments. Representative Western blot (right). For all subfigures, some error bars are too small to visualize. *p<0.05, **p≤0.01, ***p<0.001 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
9.
Figure 1

Figure 1. c-Abl/Arg inhibitors reverse doxorubicin resistance.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A) 435s/M14 melanoma and (B) BT-549 breast cancer cells were treated with doxorubicin/imatinib (72 h), and viability assessed by CellTiter-Glo. Mean±SEM for 3 independent experiments (left). Representative dose response curve (right). (C,F) Graphical representation of combination indices obtained with CalcuSyn software using dose response curves for each drug alone and in combination. >1-antagonism;  = 1-additive; <1-synergism. Graphs are representative of 3 independent experiments. (D) Cells stably expressing imatinib-resistant mutant Arg (ArgT) were transiently transfected with imatinib-resistant c-Abl (c-AblT), treated with doxorubicin/imatinib (48 h), and viability assessed. Representative experiment (left). Mean±SEM of 3 independent experiments: imatinib alone (right, top) and imatinib+doxorubicin (right, bottom). (E,F) Parental (E) and acquired doxorubicin-resistant (F) cells were drug-treated (72 h), and viability assessed. Experiments were performed ≥3 times, and representative dose response curves are shown. Mean±SEM for 3 independent experiments is shown in Figure S3B. For all subfigures, some error bars are too small to visualize. *p<0.05, **p<0.01, ***p<0.001 (see methods).

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.
10.
Figure 4

Figure 4. c-Abl inhibition decreases ABCB1 expression and activity.. From: Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-?B and HSP27/p38/AKT Pathways and by Inhibiting ABCB1.

(A) Cells were treated with imatinib (72 h), washed, treated with doxorubicin (30′) in the absence of imatinib, and intracellular doxorubicin fluorescence assessed by FACS. The experiment was performed ≥3 times; a representative experiment is shown. (B) Cells were drug- treated (72 h), and viability was assessed. Graph is representative of 3 independent experiments. Some error bars are too small to visualize. PTX = paclitaxel (30 nM), Cam = camptothecin (1 µM), Cis = cisplatin (20 µM), 5-FU = 5-fluorouracil (500 µM). (C,D) Western blot analysis. Lysate from MCF-7 cells transfected with ABCC1 served as a positive control for ABCC1 expression. Blots are representative of ≥3 independent experiments. (D) Cells were imatinib or nilotinib-treated (48 h, left) or transfected with siRNAs (right), and lysates blotted with the indicated antibodies. Mean±SEM from 3 independent experiments (below). (E) siRNA-transfected cells were treated with doxorubicin (30′), washed, and intracellular doxorubicin fluorescence assessed. Graph is representative of 3 independent experiments. Representative Western blot (right). (F, G) Cells were treated with either doxorubicin (F) or rhodamine 123 (G) in the presence of the ABCB1 inhibitor, verapamil, or imatinib for 30′, washed, and incubated with verapamil or imatinib for an additional 45′, and rhodamine 123 or doxorubicin intracellular fluorescence assessed by FACS. Graphs are representative of 3 independent experiments. *p<0.05, **p≤0.01, ***p<0.001 (see methods). n.s. = not significant.

Jonathan T. Sims, et al. PLoS One. 2013;8(1):e55509.

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