Fig. 1

DTX-P7 inhibits tumor growth and promotes tumor cells to apoptosis by favorably distributing to tumor tissues and inducing Hsp90 degradation and unfolded protein response. a Volume of xenograft tumor mass. A549 tumor-bearing mice were randomized to receive intraperitoneal injection of vehicle control, 10 mg/kg DTX-P7, 20 mg/kg DTX-P7 or 10 mg/kg DTX once a week for 4 weeks (n = 5 mice/group). Data are represented by mean ± SD. Arrows indicate the treatments. b Weight of finally dissected xenograft tumor mass. c A549 tumor-bearing mice were injected intraperitoneally with 30 mg/kg DTX or 60 mg/kg DTX-P7 followed by determination of distribution of DTX or DTX-P7 in tumor specimens throughout 72 h. DTX-P7 was quantified by free DTX released from the conjugate. d A549 cells were treated with different concentrations of DTX-P7 or 50 nM DTX-P7 for different intervals followed by total cell lysate preparation and Western blotting analysis of Hsp90 expression. e A549 cells were treated with 0, 10, 50 nM DTX-P7 or 1 nM DTX for 48 h followed by total RNA extraction and real-time PCR for analysis of Hsp90 mRNA level. f A549 cells were treated with cycloheximide (2.5 mg/mL) in the presence or absence of 50 nM DTX-P7 for various times and harvested for Western blotting analysis. Half-life of Hsp90 was determined using Image J software and plotted against treatment time. Data shown are mean ± standard deviation of three independent experiments. g–h A549 cells were treated with 50 nM DTX-P7 in the absence or presence of 10 nM bafilomycin A₁ or 20 nM bortezomib for 48 h and harvested for Western blotting analysis of Hsp90 expression in total cell lysates (g), cytosol fraction and membrane fraction (h). GAPDH and calnexin were used as loading controls for cytosol and membrane fractions, respectively. C: cytosol fraction; M: membrane fraction. i Immunohistochemistry analysis of xenograft tumor tissues for the expression of Hsp90. j Effect of DTX-P7 on unfolded protein response-related proteins in A549 cells as determined by Western blotting and the quantitative analysis of blots. β-actin was used as a loading control. k Effect of DTX-P7 on XBP1 splicing and CHOP mRNA level in A549 cells as determined by real-time PCR analysis. GAPDH was used as an internal control. l Effect of DTX-P7 on apoptosis in A549 cells by Annexin V-PI apoptotic assay. m A549 cells were incubated with 0, 10, 50 nM DTX-P7 or 1 nM DTX in the presence and absence of Z-VAD-FMK. Total cell lysates were subjected to Western blotting to assess apoptotic proteins using specific antibodies. n Hematoxylin and eosin-stained paraffin sections of tumor tissues of vehicle-, DTX- and DTX-P7-treated mice. Scale bar indicated 200 µm. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus DTX group