Fig 1.
(A) In situ liquid cell TEM image of the 10 μM Abraxane solution sample, and (B) the size statistics of the albumin-bond paclitaxel particles obtained from (A). The curve in (B) is the Gaussian fit of the data.
Fig 2.
Establishment of Abraxane-resistant NSCLC cell line.
(A) The A549/Abr cell line is resistant to Abraxane. A549 (•, black) and A549/Abr (•, red) cells were treated with the indicated increasing concentrations of Abraxane for 48 h. Cell viability was detected using MTT assay. Each point represents the mean±SD of three measurements. IC50 is the concentration of Abraxane that inhibits 50% of cell proliferation. The IC50 values for the A549 and A549/Abr cells were 11.07 nM and 1314.66 nM, respectively. (B) Growth curves of both cell lines, A549 (black dots) and A549/Abr (red dots). The curves of Abr-resistant cells rise up slowly. The doubling time of A549 cells is 49.56 h and that of A549/Abr cells is 54.65 h. (C) Morphological comparison of A549 cell line and A549/Abr cell line. Effects of Abraxane on the morphologic changes in A549 and A549/Abr cells. Cells were treated with (b and d) or without (a and c) 100 nM Abraxane for 24 h and the morphologic changes were observed by microscopy.
Table 1.
IC50 and RI values of A549 and A549/Abr cells.
Effects of selected drugs on both cell lines. RI means resistance index, ratios of IC50A549/Abr/IC50A549. Data represents mean±SD of at least three separate experiments. The results suggested multidrug-resistant characteristic of A549/Abr.
Fig 3.
(A) Number of transcripts detected as a function of read depth. X-axis: number of reads (millions) mapped to the genome; Y-axis: the number of transcripts detected in genes from the National Center for Biotechnology Information (NCBI) database. (B) Correlation between qRT-PCR and RNA-Seq for selected genes in A549, A549-100, A549/Abr and A549/Abr-100 cells. The selected genes are ABCB1, ABCC1, ABCG2, MGST1, BCL2, BAX, TP53, ANXA2, HIF1A. (C) Cluster analysis of expressed genes in four cell types. Expression intensities are displayed from green (low expression) to red (high expression).
Table 2.
GO enrichment of differentially expressed genes between A549 and A549-100.
Fig 4.
ABC transporters are significantly up-regulated in MDR cells compared with A549 parental cells.
This figure shows the expression changes between A549/Abr and A549, other comparisons are included in S3 Fig.
Table 3.
Differentially expressed genes between A549 and A549/Abr cells.
Fig 5.
Expression level of P-gp was higher in A549/Abr cells.
(A) Real-time PCR analysis of P-gp showed that mRNA level of P-gp increased in A549/Abr cells compared with A549, from 1.0 to 438.01. (B) Western blot analysis of P-gp showed that protein expression level of P-gp increased in A549/Abr cells compared with A549, 85.41 and 0.45 respectively as highlighted with ** versus *. The change is significant (P<0.01) by Student’s t-test.
Fig 6.
P-gp up-regulation mediates Abraxane resistance in lung cancer cell line.
(A) The affection of a P-gp inhibitor verapamil (VP) used alone on A549 and A549/Abr cell viabilities. Both cells were treated with indicated increasing concentrations of VP for 48 h. Cell viability was detected using MTT assay. VP itself didn’t affect much on the viability of A549 and A549/Abr cells. (B-C) Both A549 and A549/Abr cells were treated with indicated increasing concentrations of Abraxane for 48 h at the presence of 4 μg/mL VP. IC50 of Abraxane for A549 cells (B) almost remained the same, while that of A549/Abr cells (C) significantly decreased after VP treatment. (D) Comparision of A549/Abr cell viabilities when treated with VP (4 μg/mL) alone, Abraxane (100 nM) alone or 4 μg/mL VP together with 100 nM Abraxane.