1,25D3 enhances antitumor activity of gemcitabine and cisplatin in human bladder cancer models

Abstract

Background: 1,25 dihydroxyvitamin D3 (1,25D3) potentiates the cytotoxic effects of several common chemotherapeutic agents. The combination of gemcitabine and cisplatin is a current standard chemotherapy regimen for bladder cancer. The authors investigated whether 1,25D3 could enhance the antitumor activity of gemcitabine and cisplatin in bladder cancer model systems.

Methods: Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D3 followed by gemcitabine and cisplatin. Apoptosis was assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate-based caspase activity assay. The cytotoxic effects were examined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and in vitro clonogenic assay. p73 protein levels were assessed by immunoblot analysis. Knockdown of p73 was achieved by siRNA. The in vivo antitumor activity was assessed by in vivo excision clonogenic assay and tumor regrowth delay in the T24 xenograft model.

Results: 1,25D3 pretreatment enhanced gemcitabine and cisplatin-induced apoptosis and the activities of caspases 8, 9, and 3 in T24 and UMUC3 cells. 1,25D3 synergistically reduced gemcitabine and cisplatin-suppressed surviving fraction in T24 cells. 1,25D3, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by gemcitabine and cisplatin or 1,25D3 and gemcitabine and cisplatin. p73 expression was lower in human primary bladder tumor tissue compared with adjacent normal tissue. Knockdown of p73 increased clonogenic capacity of T24 cells treated with 1,25D3, gemcitabine and cisplatin, or 1,25D3 and gemcitabine and cisplatin. 1,25D3 and gemcitabine and cisplatin combination enhanced tumor regression compared with 1,25D3 or gemcitabine and cisplatin alone.

Conclusions: 1,25D3 potentiates gemcitabine and cisplatin-mediated growth inhibition in human bladder cancer models in vitro and in vivo, which involves p73 induction and apoptosis.

FIGURE 1

VDR expression in bladder cancer cell lines. Human bladder cancer cell lines T24 or UMUC3 were treated with vehicle control ETOH, 375 nM or 1500 nM 1,25D₃ for 72 h. VDR expression was assessed by immunoblot analysis. Actin was the loading control. Results are representative of three independent experiments.

FIGURE 2

1,25D₃ enhances GC-induced apoptosis. (a) T24 cells were treated with ETOH or 1.5 μM 1,25D₃ for 24 h, followed by 25 nM gemcitabine (Gem) and 0.75 μg/ml cisplatin (cDDP) for 48 h. Apoptosis was assessed by annexin V-PE staining by flow cytometry. The populations of annexin V−/7AAD−, annexin V+/7AAD− and annexin V+/7AAD+ corresponded to live cells, early apoptotic cells, and late apoptotic cells. The results were summarized in a bar graph as mean ± SD of annexin V positive cells in triplicate experiments and are representative of two independent experiments. (b) UMUC3 cells were treated with ETOH or 1.5 μM 1,25D₃ for 24 h, followed by 0.15 μg/ml cisplatin and 100 nM gemcitabine for 48 h. Apoptosis was assessed by annexin V-PE staining by flow cytometry. The results were summarized in a bar graph as mean ± SD of annexin V positive cells in triplicate experiments and are representative of two independent experiments.

FIGURE 3

1,25D₃ enhances GC-induced caspase activity. (a) T24 cells were treated with ETOH or 1.5 μM 1,25D₃ for 24 h, followed by 25 nM gemcitabine (Gem) and 0.75 μg/ml cisplatin (cDDP) for 48 h. UMUC3 cells were treated with ETOH or 1.5 μM 1,25D₃ for 24 h, followed by 100 nM Gem and 0.15 μg/ml cDDP for 48 h. The cleavages of caspases-8, 10, 9, 3 and PARP were evaluated by immunoblot analysis. Actin was the loading control. Results are representative of three independent experiments. (b–d) T24 cells were treated with ETOH or 1.5μM 1,25D₃ for 24 h, followed by 25 nM Gem and 0.75μg/ml cDDP for 48 h. The activities of caspases-8, 9 and 3 were examined by substrate-based caspase activity assays. Results are the mean ± SD of triplicate experiments and are representative of three independent experiments.

FIGURE 4

Antitumor effect and interaction between 1,25D₃ and GC. (a) T24 cells were pretreated with varying doses of 1,25D₃ for 24 h followed by differing concentrations of GC with a fixed ratio of 1:100 for 48 h and assayed by MTT assay. Fraction affected (Fa) was calculated as 1-(MTT value of the treatment cells)/(MTT value of control ETOH-treated cells). (b–c) T24 cells were treated as in 4a with more focused doses of 1,25D₃ and the same doses of GC. Combination index (CI) values for the different combination treatment (b) or for the Fa (c) were determined using CalcuSyn software. A CI < 1 denotes synergy. (d) T24 cells were treated with ETOH or 1 μM 1,25D₃ for 24 h followed by 1.25 nM gemcitabine and 0.1 μg/ml cisplatin and subjected to in vitro clonogenic assay. The clones were fixed, stained and counted on day 9. Results are the mean ± SD of triplicate experiments and are representative of three independent experiments.

FIGURE 5

p73 plays a role in 1,25D₃ and GC-mediated growth inhibition. (a) T24 cells were treated with ETOH or 1.5 μM 1,25D₃ for 24 h followed by 25 nM gemcitabine and 0.75μg/ml cisplatin for 48 h. UMUC3 cells were treated with ETOH vehicle control or 1.5μM 1,25D₃ for 24 h followed by 100 nM Gem and 0.15μg/ml cDDP for 48 h. The levels of p73 were assessed by immunoblot analysis. Actin was the loading control. Results are representative of three independent experiments. (b) Four pairs of primary human bladder tumors or adjacent normal tissue were homogenized and the levels of p73 were assessed by immunoblot analysis. A nonspecific band was presented as the loading control. (c) T24 cells were transfected with 50 nM of siRNA-p73 or a non-specific (NS) siRNA for 48 h. The levels of p73 were assessed by immunoblot analysis. Actin was the loading control. Results are representative of three independent experiments. (d) siRNA-transfected T24 cells were treated with ETOH or 1 μM 1,25D₃ for 24 h, followed by 1.25 nM gemcitabine and 0.1 μg/ml cisplatin and subjected to in vitro clonogenic assay. Results are the mean ± SD of triplicate experiments and are representative of three independent experiments.

FIGURE 6

1,25D₃ potentiates the antitumor activity of GC in vivo. (a) Nude mice bearing palpable T24 tumors were treated with saline, 1,25D₃ (0.625 μg/mouse daily for 3 d), gemcitabine (6 mg/mouse on day 2) and cisplatin (0.12 mg/mouse on day 3), or the combination of 1,25D3 and GC. The tumors were harvested 24 h after the last treatment and in vivo excision clonogenic assay was performed. (b) Tumor growth was monitored and measurements taken on the days indicated. Tumor volumes were calculated by the following formula: volume = (length × width2)/2. For each tumor, fractional tumor volumes were calculated using the following formula: Fractional tumor volume = (volume on day measured)/(initial tumor volume). *, P < .05, 1,25D₃ and GC vs. GC.