Fig 2: ABCB1 is essential to chemoresistance in breast cancer. (A) Expression of ABCB1 in breast cancer samples collected pre- and postchemotherapy in the dataset GSE87455. (B) Representative images of IHC staining for ABCB1 in chemoresistant and chemosensitive breast cancer samples. (C) Correlation between the pCR rate and ABCB1 expression in breast cancer samples obtained from 67 patients. (D, E) Protein (D) and mRNA (E) levels of ABCB1 in MCF7 and MCF7/DOX cells determined by Western blotting and qPCR. (F, G) Protein (F) and mRNA (G) levels of ABCB1 in MCF7 cells transfected with an empty vector or ABCB1 vector determined by Western blotting and qPCR. (H) Survival rate and IC50 of MCF7 cells transfected with an empty vector or a STAT5a vector after treatment with DOX for 48 h determined by a CCK8 assay. (I) Representative images and quantification of colonies formed by MCF7 cells transfected with the empty vector or ABCB1 vector in medium containing DOX. (J, K) Protein (J) and mRNA (K) levels of ABCB1 in MCF7/DOX cells transfected with scramble siRNA or ABCB1-targeting siRNA detected by Western blotting and qPCR. (L) Survival rate and IC50 of MCF7/DOX cells transfected with scramble siRNA or ABCB1-targeting siRNA after treatment with DOX for 48 h determined by a CCK8 assay. (M) Representative images and quantification of colonies formed by MCF7/DOX cells transfected with scramble siRNA or ABCB1-targeting siRNA in medium containing DOX. (N, O) Expression of apoptosis markers in MCF7 cells transfected with the empty vector or ABCB1 vector (N) and in MCF7/DOX cells transfected with scramble siRNA or ABCB1-targeting siRNA in the presence of DOX (O). (P, Q) Representative images (P) and quantification (Q) of the accumulation of DOX in MCF7 and MCF7/DOX cells after treatment with DOX. (R, S) Representative images (R) and quantification (S) of the accumulation of DOX in MCF7/DOX cells transfected with scramble siRNA or ABCB1-targeting siRNA after DOX treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 3: DHW-221 has no obvious toxicity in DHW-221-administered nude mice. (A) HE staining of the heart, liver, kidney, and speen from orthotopic nude mice. Scale bar = 100 µm. (B) The viscera index of organs (heart, liver, speen, lung, and kidney) and the biochemical parameters of heart (CK), liver (ALT, AST), and kidney (CRE) from the serum of each group in orthotopic nude mice. Data were presented as mean ± SD. Statistical comparisons were carried out with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons (n = 6). (C) Molecular mechanism underlying the anticancer effects of DHW-221 on the P-gp and PI3K/Akt/FOXO3a signaling pathway in A549/Taxol cells.

Fig 4: DHW-221 accelerates intracellular Rho-123 accumulations and inhibits P-gp expression by binding to P-gp. (A) Comparison of P-gp in A549 and A549/Taxol cells. Statistical comparisons were performed with unpaired Student’s t-test (n = 3). **p < 0.01 versus A549. (B, C) After treatment of DHW-221 or verapamil for 48 h, Rho-123 (15 µM) was added into the mediums to co-incubate with DHW-221 or verapamil for 3 h in dark. The intracellular accumulation of Rho-123 was evaluated by flow cytometry and confocal microscope, respectively. Verapamil (VRP, 10 μM) was used as a positive control. Green fluorescence indicated Rho-123, and blue fluorescence indicated DAPI. Scale bar = 100 μm. Nuclear mean fluorescence intensity of Rho 123 was measured by ImageJ and analyzed using FlowJo 10 software. Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus control. (D) Effect of DHW-221 or verapamil to reverse Taxol resistance for 48 h in A549/Taxol cells. (E) Western blot analysis of DHW-221 inhibited P-gp expression in A549/Taxol cells. (F) Ribbon diagram of human ABCB1 (PDB code: 7A6E, purple spiral) bound to drug (membrane region) and predicted binding modes for DHW-221 (blue stick) and tariquidar (orange stick, a known third-generation P-gp inhibitor) with ABCB1. Hydrogen bonds were shown as green dashed lines. Key residues for DHW-221 and tariquidar interaction were highlighted. (G) Effect of DHW-221 on the thermal stability of ABCB1 was quantitatively detected by a cellular thermal shift assay (CETSA). Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons (n = 3). Data are presented as mean ± SD. **p < 0.01, ****p < 0.0001versus control.

Fig 5: Characterization of the vasculature of intracranial tumor modelsThe leakiness of the vasculature of intracranial GBM8, U87, Mel57, and Mel57VEGF tumors was assessed by T2-weighted, T1-weighted pre-contrast and T1-weighted post-gadolinium (Gd) contrast magnetic resonance imaging, by autoradiography following intravenous (i.v.) administration of 14C-aminoisobutyrate (AIB) and by fluorescence microscopy following i.v. Texas Red (TxRed). Histochemical hematoxylin and eosin (H&E) staining was used as a reference for tumor location. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) expression was demonstrated by immunohistochemistry. All of the microscopy images are representative of n = 5–13 animals. Scale bars, 100 μm.See also Figures S1 and S2.