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. 2014 Dec 21;14(1):142.
doi: 10.1186/s12935-014-0142-4. eCollection 2014.

Differential drug resistance acquisition to doxorubicin and paclitaxel in breast cancer cells

Feifei Xu #  1 , Fengliang Wang #  2 , Ting Yang  1 , Yuan Sheng  1 , Ting Zhong  1 , Yun Chen  1
Affiliations

Differential drug resistance acquisition to doxorubicin and paclitaxel in breast cancer cells

Feifei Xu et al. Cancer Cell Int. .

Abstract

Background: Several signal transduction pathways have been reported being involved in the acquisition of P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) upon exposure to anti-cancer drugs, whereas there is evidence indicating that the expression and activity of P-gp were not equally or even reversely modulated by different drugs.

Methods: To further illustrate this drug-specific effect, possible mechanisms that enable breast cancer cells MCF-7 to acquire MDR to either paclitaxel (PTX) or doxorubicin (DOX) were investigated in a time-dependent manner.

Results: The results suggested that at least two pathways participated in this process. One was the short and transient activation of NF-κB, the second one was the relatively prolonged induction of PXR. Both PXR and NF-κB pathways took part in the PTX drug resistance acquisition, whereas DOX did not exert a significant effect on the PXR-mediated induction of P-gp. Furthermore, the property of NF-κB activation shared by DOX and PTX was not identical. An attempt made in the present study demonstrated that the acquired resistance to DOX was via or partially via NF-κB activation but not its upstream receptor TLR4, while PTX can induce the drug resistance via TLR4-NF-κB pathway.

Conclusions: To our knowledge, this report is among the first to directly compare the time dependence of NF-κB and PXR pathways. The current study provides useful insight into the distinct ability of DOX and PTX to induce P-gp mediated MDR in breast cancer. Different strategies may be required to circumvent MDR in the presence of different anti-cancer drugs.

Keywords: Doxorubicin (DOX); Drug-specific; Multi-drug Resistance Acquisition (MDR); P-glycoprotein (P-gp); Paclitaxel (PTX).

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Figures

Figure 1
Figure 1
Representative Western blotting (A) and quantitative analysis (B) of P-gp, PXR and NF-κB p65, and mRNA of MDR1 determined by RT-PCR (C). GAPDH was used as the internal control.
Figure 2
Figure 2
The impact of DOX and PTX on P-gp expression and activity. (A) The concentration-dependent effect of DOX and PTX on mRNA level of MDR1. The cells were treated with drugs for 12 h. (B) The time-dependent effect of drugs on mRNA levels of MDR1 with 100 nM DOX/25 nM PTX treatment. (C) Rho-123 fluorescence ratio in MCF-7 cells with DOX and PTX treatments at different concentrations. (D) Rho-123 fluorescence ratio in MCF-7 cells with 100 nM DOX/25 nM PTX treatment for 0, 2, 4, 8, 12, 24, 36, 48 h. (E) MDR1 mRNA levels in MCF-7 cells with 8 h/36 h DOX and PTX treatment. *, p < 0.05; **, p < 0.01 (n = 3). (F) Rho-123 fluorescence ratio in MCF-7 cells with 8 h/36 h DOX and PTX treatment. *, p < 0.05 (n = 3).
Figure 3
Figure 3
p65 translocation in MCF-7 cells after the treatment of DOX and PTX. (A) Western blotting and (B) nuclear:cytoplasmic ratio of NF-κB p65 in cytoplasmic and nuclear fractions of DOX and PTX treated MCF-7 cells at 0, 0.5, 1, 2, 4, 8 h. (C) Western blotting of NF-κB p65 after 2 h treatment of DOX and PTX using Actin and TBP as loading controls. (D) A quantitative analysis of nuclear:cytoplasmic ratio of p65 in (C). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3).
Figure 4
Figure 4
Effect of NF-κB inhibitor Bay 11-7082 in DOX and PTX treated cells. (A-B) Effect of Bay 11–7082 on the expression of NF-κB p65 in cytoplasmic and nuclear fractions with 8 h treatment of DOX and PTX and Bay 11–7082 for 2 h. (C) Relative MDR1 mRNA of DOX and PTX treated cells. *, p < 0.05; **, p < 0.01, blank P > 0.05 (n = 3). (D) Rho-123 fluorescence ratio of DOX and PTX treated cells. *, p < 0.05; **, p < 0.01, blank, P > 0.05 (n = 3).
Figure 5
Figure 5
Effect of anti-TLR4 antibody. (A-B) Western blotting and quantitative analysis of nuclear and cytoplasmic p65 after the use of TLR4-blocking antibody. (C) Relative MDR1 mRNA level and Rho-123 fluorescence ratio of DOX and PTX treated cells. *, p < 0.05; **, p < 0.01, blank, p > 0.05 (n = 3).
Figure 6
Figure 6
Confocal images of p65 in cells after the treatment of DOX (A) and PTX (B) with or without Bay 11–7082 inhibition and TLR4 antibody blocking. Cells were stained with Dylight 488 (green fluorescence) for p65 and co-stained with DAPI (blue) for positive identification of nuclei. The cells (arrow) are also enlarged at single-cell level for clarification (right images).
Figure 7
Figure 7
Impact of PXR activation in DOX and PTX treated cells. (A) PXR mRNA expression with the treatment of DOX and PTX at 0, 4, 8, 12, 24, 36, 48 h. (B) Western blot analysis of PXR with 24 h treatment of DOX and PTX. **, p < 0.01 (n = 3). (C) RT-PCR analysis of PXR mRNA in PTX only (PTX) or treated cells exposed to transfection conditions in the absence of siRNA (mock) or cells transfected with non-targeting (NC) or PXR-specific siRNA (siRNA). (D) Comparison of MDR1 mRNA expression in the cells exposed to transfection conditions with 8 h/36 h DOX and PTX treatment. **, p < 0.01 (n = 3). (E) Rho-123 fluorescence ratio in the cells exposed to transfection conditions with 8 h/36 h DOX and PTX treatment. *, p < 0.05 (n = 3).
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