1alpha,25-Dihydroxyvitamin D3 potentiates cisplatin antitumor activity by p73 induction in a squamous cell carcinoma model

Abstract

1alpha,25-Dihydroxyvitamin D3 (1,25D3) exhibits antitumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 before cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 before cisplatin compared with either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin but increased by 1,25D3 in SCC cells. Knocking down p73 by small interfering RNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin antitumor activity in vitro and in vivo in a SCC model system possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment.

Figure 1

SCC-DR cells are resistant to 1,25D₃ treatment. A, SCC or SCC-DR cells were treated with 0, 10, or 500 nM of 1,25D₃ and subjected to in vitro clonogenic assay. After staining, colonies were viewed and counted on a light microscope and photographed. Surviving fraction was calculated by dividing the cloning capacity of treated cells to that of ETOH control. Results of the surviving fraction are the mean ± SD of triplicate experiments and are representative of two independent experiments. B, SCC and SCC-DR cells were treated with 0 to 1000 nM of 1,25D₃ for 48 h, and the levels of VDR and caspase 3 were assessed by immunoblot analysis. Actin was the loading control. Results are representative of two independent experiments. (c) SCC-DR cells were treated with 10 nM of 1,25D₃ for 5 to 240 min, and the levels of phosphorylated Akt and ERK1/2 were evaluated by immunoblot analysis. Total Akt or ERK1/2 level was assessed as the loading control.

Figure 2

1,25D₃ sensitizes SCC cells to cisplatin treatment. A, SCC or SCC-DR cells were treated with 0 to 1 μg/ml of cisplatin for 48 h, and cell viability was assessed by Trypan blue exclusion assay. Results are the mean ± SD of triplicate experiments and are representative of two independent experiments. B, SCC or SCC-DR cells were pretreated with vehicle control ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h. Cells were harvested after an additional 48 h of incubation. Cytotoxicity was examined by LDH Cytotoxicity Detection Kit. C, Various dilutions of SCC or SCC-DR cells were plated in six-well tissue culture plates over night. They were pretreated with ETOH or 10 nM 1,25D₃ for 24 h, and then incubated without further treatment or 0.5 μg/ml cDDP for 2 h and subjected to in vitro clonogenic assay. Results are representative of two to three independent experiments. cDDP, cisplatin. *, P < 0.00001, vs. ETOH; #, P < 0.01, vs. cDDP.

Figure 3

1,25D₃ promotes cisplatin anti-tumor activity in SCC in vivo. A, SCC tumor- bearing mice (3 to 5 per group) were treated with saline or 0.625 μg of 1,25D₃ daily for 3 d. On day 3, mice also received 3 mg/kg of cisplatin. Both agents were administered i.p. The in vivo excision clonogenic assay was performed 24 h after the last treatment. Each point represents the mean surviving fraction for total clonogenic cells per gram of tumor. *, P < 0.001, **, P < 0.0001, vs. saline; #, P < 0.01, vs. saline. B, C3H mice (10 per group) bearing palpable subcutaneous SCC tumors were treated with either saline, 0.25 μg of 1,25D₃ daily for 3 d, 6 mg/kg of cisplatin on day 3, or the combination of 0.25 μg of 1,25D₃ daily for 3 d and 6 mg/kg of cisplatin on day 3. Both agents were administered i.p. Tumor measurements were obtained on the days indicated, and fractional tumor volumes were calculated as described in Methods. Data points represent the mean ± SD fractional tumor volume for 10 mice/group. *, P < 0.05. cDDP, cisplatin.

Figure 4

1,25D₃ promotes cisplatin to induce apoptosis. A, SCC cells were pretreated with ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h and an additional 48 h of incubation. Cells were harvested, lysed, and DNA fragmentation was evaluated by Cell Death Detection ELISA^PLUS according to the manufacturer’s protocol. The enrichment factor was used as a parameter of apoptosis and shown on the y axis as mean ± SD of triplicate experiments. B, SCC or SCC-DR cells were pretreated with vehicle control ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h. After an additional 48 h of incubation, cells were harvested and pro-caspases 8, 9, 10, 3 and PARP levels were evaluated by immunoblot analysis. Actin was the loading control. Results are representative of three independent experiments. cDDP, cisplatin.

Figure 5

1,25D₃-incrased p73 protein level contributes to cisplatin-induced growth inhibition. A, SCC or SCC-DR cells were pretreated with ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h and an additional 48 h of incubation. Cells were harvested and p53, p63 and p73 levels were evaluated by immunoblot analysis. Actin was the loading control. B, SCC cells were transfected with siRNA-NS, siRNA- p73, or left untransfected for 24 h, followed by the treatment with 1,25D₃ for 48 h. p73 mRNA level was evaluated by Real-time quantitative PCR. Results are the mean ± SD of the relative expression level to GAPDH. NS, non-specific; *, undetectable. Following siRNA-transfection, SCC cells were pretreated with ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h. Anti-proliferative effect was assessed by in vitro clonogenic assay. Results are representative of two independent experiments. C, SCC cells were transfected with siRNA-NS, siRNA-UFD2a, or left untransfected for 24 h, followed by the treatment with 1,25D₃ for 48 h. p73 protein level was evaluated by immunoblot analysis. Actin was the loading control. UFD, UFD2a. Following siRNA- transfection, SCC cells were pretreated with ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h. Anti-proliferative effect was assessed by in vitro clonogenic assay. Results are representative of two independent experiments. cDDP, cisplatin.

Figure 6

A, p73 contributes to 1,25D₃ and cisplatin-induced apoptosis. SCC cells were transfected with siRNA-NS, siRNA-p73, or left untransfected for 24 h. Cells were then pretreated with ETOH or 10 nM 1,25D₃ for 24 h followed by 0.5 μg/ml of cisplatin for 2 h and an additional 48 h of incubation. Cells were harvested and DNA fragmentation was evaluated by Cell Death Detection ELISA^PLUS kit. The enrichment factor was used as a parameter of apoptosis and shown on the y axis as mean ± SD of triplicate experiments and are representative of three independent experiments. cDDP, cisplatin. B, A schematic presentation of the 1,25D₃ potentiation of cisplatin anti-tumor activity. SCC cells are resistant to cisplatin treatment. 1,25D₃ induces p73 accumulation and sensitizes SCC cells to cisplatin-mediated growth inhibition through caspase 8/10-caspase 3-dependent apoptotic pathway.