Intravesical combination treatment with antisense oligonucleotides targeting heat shock protein-27 and HTI-286 as a novel strategy for high-grade bladder cancer

Suppression of Hsp27 protein enhances cytotoxicity in bladder cancer cells. A, sequence-specific suppression of Hsp27 protein levels by OGX427 in bladder cancer cells. Cells were treated with 50 nmol/L OGX427 or ScrB control oligodeoxynucleotide for 2 consecutive days; 72 h later, total protein was extracted and Hsp27 expression was analyzed by immunoblotting. B, flow cytometric analysis after treatment with OGX427and HTI-286. KU7-luc cells were treated with 50 nmol/L OGX427 or control oligodeoxynucleotide daily for 2 d. After 72 h of incubation with medium ± 0.6 nmol/L HTI-286, flow cytometry was used to quantify the percentage of cells in each cell cycle phase. *, P < 0.05. C, i, RT-PCR analysis showing MDR-1 expression in four different bladder cancer cell lines; ii, effects of OGX427 on HTI-286–induced cytotoxicity in bladder cancer cells. Cells were transfected with 50 nmol/L OGX427 or ScrB control oligodeoxynucleotide for 2 consecutive days and incubated with medium ± 0.5 nmol/L HTI-286 for 72 h. Cell viability was determined by MTS assays and is indicated by the ratio to cell viability when treated with drug vehicle only. *, P < 0.05. D, enhancement of various chemotherapeutic agents–induced cytotoxicity by OGX427 in KU7-luc cells. Cells were transfected with 50 nmol/L OGX427 or ScrB control oligodeoxynucleotide for 2 consecutive days and incubated with medium ± 20 nmol/L paclitaxel, 7.5 μmol/L cisplatinum (CDDP), or 10 nmol/L gemcitabine for 48 to 72 h. Cell viability was determined by MTS assays. *, P < 0.05.

Suppression of Hsp27 combined with chemotherapeutic agents inactivated Akt signaling and promoted apoptosis. A, Western blot analysis showing the effects of combination treatment with OGX427 and HTI-286 in KU7-luc cells. Cells were treated as described in Fig. 2B and then subjected to Western blot analysis. Black arrows, cleaved forms of caspase-9 and poly(ADP-ribose) polymerase (PARP). B, effects of p38 inhibition on the susceptibility to HTI-286 and OGX427 of KU7-luc cells. Cells were transfected with 50 nmol/L OGX427 or ScrB control oligodeoxynucleotide for 2 consecutive days and incubated with medium ± 0.5 nmol/L HTI-286 and/or 10 μmol/L SB203580 for 72 h and then subjected to Western blot analysis. C, Western blot analysis showing the effects of combination treatment with OGX427 and HTI-286 in KK47 cells. Cells were treated as described in Fig. 2B and then subjected to Western blot analysis. D, Western blot analysis showing the effects of combination treatment with OGX427 and various chemotherapeutic agents in KU7-luc cells. Cells were treated as described in Fig. 2D and then subjected to Western blot analysis.

Overexpression of Hsp27 protein contributes to chemoresistance against HTI-286 in bladder cancer cells. A, top, expression level of Hsp27 in lysates harvested from T24 cells infected with empty lentiviral vector (mock) or Hsp27-expressing lentiviral vector (Hsp27). T24 cells were infected with lentivirus as described in Materials and Methods, and their cell lysates were subjected to Western blot analysis. Bottom, effect of overexpression of Hsp27 protein on the susceptibility to HTI-286 in T24 cells. The results of MTS assays of T24 mock (open circles) and T24 Hsp27 (black squares) treated with HTI-286 at the indicated concentrations for 72 h are shown as average ± SE from three independent experiments. Cell viability is indicated as the ratio to cell viability with no treatment. *, P < 0.05. B, induction of Akt activation and reduced poly(ADP-ribose) polymerase cleavage after HTI-286 treatment by Hsp27 overexpression. Cells were treated by 1.0 nmol/L HTI-286 or DMSO for 72 h and then lysed in radioimmunoprecipitation assay buffer and subjected to Western blot analysis. C, effects of Akt inactivation on the susceptibility to HTI-286 of T24 mock cells and T24 Hsp27 cells. Cells were treated by 1.0 nmol/L HTI-286 (white column) or HTI-286 + 20 μmol/L LY294002 (black column) for 72 h. Cell viability was determined by MTS assays and is indicated by the ratio to cell viability when treated with drug vehicle only. *, P < 0.05.

Intravesical instillation of HTI-286 and OGX427 suppresses the in vivo tumor growth on orthotopic bladder cancer xenografts. A, 28 female nude mice were inoculated with 2 × 10⁶ KU7-luc tumor cells on day 0 and randomized on day 5 to receive either treatment with ScrB + DMSO, ScrB + HTI-286, OGX427 + DMSO, or OGX427 + HTI-286. ScrB or OGX427 was administered three times a week, and HTI-286 or DMSO was administered biweekly for 2 consecutive weeks. Tumor growth was determined on days 5, 10, 15, 20, and 25 in a Xenogen IVIS200 camera. *, P < 0.05, Kruskal Wallis test; †, P < 0.05, between ScrB + HTI-286 and OGX427 + HTI-286 groups; §, P < 0.01, between OGX427 + DMSO and OGX427 + HTI-286 groups, Mann-Whitney U tests. B, representative sequences of bioluminescence images of mice from each treatment group taken on the day of randomization (day 5) and at days 15 and 25.