Figure 1.
Workflow for the comparative secretome analysis of MCF-7 and MCF-7/Dox.
Illustration of the label-free approach to identify the differential proteins in the CM of MCF-7 and MCF-7/Dox cell lines.
Figure 2.
Overview of proteins identified in the CM of MCF-7 and MCF-7/Dox and their subcellular localization.
Overlap of total proteins (A) and secreted proteins (B) identified in the CM of MCF-7 and MCF-7/Dox. (C) Subcellular localization of total proteins identified in the CM of MCF-7 and MCF-7/Dox. (D, E) Subcellular localization of proteins identified in the CM of MCF-7 and MCF-7/Dox, respectively.
Figure 3.
Functional analysis and classification of DESPs.
(A) The functional analysis of DESPs between MCF-7 and MCF-7/Dox. (B) The classification of DESPs between MCF-7 and MCF-7/Dox.
Table 1.
List of the ECM related DESPs.
Table 2.
List of growth factors among the DESPs.
Table 3.
List of cytokines among the DESPs.
Table 4.
List of the remained DESPs.
Figure 4.
Canonical pathways analysis of DESPs with potential dual functions in drug resistance and tumor metastasis.
32 DESPs with dual functions were analyzed by software IPA. The top 10 canonical pathways were mapped onto the DESPs, such as IGF-1 signaling, acute phase response signaling and IL-6 signaling etc. IL-6 and IL-18 were located as centers to link multiple signaling pathways.
Figure 5.
Validation of the expression level of IL-18 and its role in drug resistance.
(A) The mRNA level of IL-18 in MCF-7, MCF-7/Dox and MCF-7/DoxH. The amount of IL-18 mRNA was normalized against the expression of GAPDH and the data were presented as mean ± SD (n = 3). **, p<0.01. (B) The concentration of IL-18 in the CM of MCF-7, MCF-7/Dox and MCF-7/DoxH. The data were presented as mean ± SD (n = 3). **, p<0.01. (C) The expression level of IL-18 in the lysates of 18 breast tumor tissues, including 12 drug-sensitive tissues and 6 drug-resistant tissues. The data were presented as mean ± SD (n = 3). **, p<0.01. (D) The doxorubicin dose dependent survival rate of MCF-7 in the absence or presence of 40 ng/mL rIL-18. Data were presented as mean ± SD (n = 3). **, p<0.01. (E) The effect of anti-IL-18 neutralization on the survival rate of MCF-7/Dox. Data were presented as mean ± SD (n = 3). *, p<0.05, **, p<0.01.