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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Cancer. 2010 Jul 1;116(13):3294–3303. doi: 10.1002/cncr.25059

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

Yingyu Ma 1, Wei-Dong Yu 1, Donald L Trump 2, Candace S Johnson 1
PMCID: PMC2891990  NIHMSID: NIHMS189714  PMID: 20564622

Abstract

Background

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

Methods

Human bladder cancer T24 and UMUC3 cells were pretreated with 1,25D3 followed by GC. Apoptosis were assessed by annexin V staining. Caspase activation was examined by immunoblot analysis and substrate-based caspase activity assay. The cytotoxic effects were examined using 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 GC-induced apoptosis and the activities of caspases- 8, 9 and 3 in T24 and UMUC3 cells. 1,25D3 synergistically reduced GC-suppressed surviving fraction in T24 cells. 1,25D3, gemcitabine, or cisplatin induced p73 accumulation, which was enhanced by GC or 1,25D3 and GC. 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, GC or 1,25D3 and GC. 1,25D3 and GC combination enhanced tumor regression compared with 1,25D3 or GC alone.

Conclusions

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

Keywords: 1,25D3; gemcitabine; cisplatin; p73; bladder cancer

INTRODUCTION

Bladder cancer is the fourth most common cancer diagnosed and eighth leading cause of cancer death in men. In 2009, there are estimated 70,980 new cases of bladder cancer diagnosed in the USA and 14,330 individuals died of bladder cancer.1 Combination treatment with gemcitabine and cisplatin (GC) is the current standard chemotherapy regimen for locally advanced and metastatic bladder cancer.2, 3 Although GC regimen is active in bladder cancer treatment, limited response rate and drug resistance remain major clinical problems. Therefore, new approaches to treatment of bladder cancer are desirable.

Cisplatin (cis-diammine-dichloro-platinum (II), cDDP) is a DNA damaging agent that forms platinum-DNA adducts and leads to apoptosis in tumor cells.4 Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) is a synthetic pyrimidine nucleoside analogue that has structural and metabolic similarities with deoxycytidine and cytosine arabinoside.5 Its active metabolites cause cytotoxicity by inhibiting DNA polymerase and being incorporated into DNA and thereby inhibiting DNA synthesis and inducing apoptosis.6

Both cisplatin and gemcitabine have been reported to increase the level of p73 protein which contributes to drug-mediated apoptosis in human colon cancer cells.7, 8 As a p53 homologue, p73 is involved in cancer development. p73 gene encodes two major groups of isoforms with opposite functions. The isoforms containing the transactivation domain (TAp73) are pro-apoptotic and the NH2-terminal truncated isoforms (ΔNp73) are anti-apoptotic.9 TAp73−/− mice developed spontaneous tumors, most frequently lung adenocarcinomas, and they were more sensitive to carcinogens.10 These results demonstrated that TAp73 had tumor suppressor activity.10 The loss of TAp73α protein expression has been associated with advanced tumor stage in bladder cancer.11

1α, 25-dihydroxyvitamin D3 (1,25D3), the most active vitamin D metabolite, has broad spectrum antitumor activities in vitro and in vivo.12 The antitumor effects of 1,25D3 are achieved through a number of mechanisms: inducing cell cycle arrest, apoptosis, cancer cell differentiation and suppressing angiogenesis.12 Epidemiological and experimental studies support a role of 1,25D3 in cancer prevention and treatment.13, 14 Low levels of plasma vitamin D are associated with higher cancer incidence and mortality in men in colorectal, breast, lung and prostate cancers.15–18 In addition, 1,25D3 potentiates the antitumor effects of common chemotherapeutic agents such as paclitaxel, cisplatin, carboplatin and doxorubicin.12 Clinical trials are underway to evaluate the role of 1,25D3, alone or in combination with other chemotherapeutic agents, in the treatment of several solid tumors.12

In this study, we investigate the role of 1,25D3 pretreatment in potentiating the antitumor activity of GC in human bladder cancer, in vitro and in vivo, and the potential role of p73.

MATERIALS AND METHODS

Materials

1,25D3 was from Hoffmann-LaRoche (Nutley, NJ). Gemcitabine (Gemzar) was from Eli Lilly and Company (Indianapolis, IN). Cisplatin (Platinol-AQ) was obtained from Bristo-Myers Squibb Company (Princeton, NJ). Anti-VDR (sc-1008) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase 3 (9662), anti-caspase 8 (4927), anti-caspase 9 (9504), anti-caspase 10 (9752) and cleaved PARP (9541) were from Cell Signaling Technology (Beverly, MA). Anti-p73 (IMG-246, clone 5B429) was from Imgenex (San Diago, CA). Anti-actin (CP-01) was from Calbiochem (San Diego, CA).

Cell culture and tumor model systems

Human bladder cancer cell lines T24 and UMUC3 were cultured in McCoy’s 5A media supplemented with 10% FBS and 1% penicillin/streptomycin sulfate. The mice protocols used for in vivo excision clonogenic assays and tumor regrowth delay were approved by the Institutional Animal Care and Use Committee at Roswell Park Cancer Institute.

Apoptosis assay

T24 and UMUC3 cells were collected and stained with PE conjugated annexin V according to manufacturer’s instructions as described previously.19

Immunoblot analysis

T24 and UMUC3 cells were treated as indicated, harvested and lysates prepared as previously described.19 Four pairs of primary human bladder tumors or adjacent normal tissue were homogenized in lysis buffer using a homogenizer (Brinkmann Polytron PT 10/35). Immunoblot analysis was performed as described.19

Caspase activity assays

The activities of caspases-3, 8, and 9 were measured using the Caspase-family Colorimetric Assay kit from R&D Systems (Minneapolis, MN) following the manufacturer’s protocol. Caspase activity was assessed by absorbance divided by protein loading.

In vitro cytotoxicity assays and dose-effect analysis

T24 cells were plated in 96-well tissue culture plates. Cells were treated with ethanol (ETOH) or pretreated with varying concentrations (0.02–6 μM) of 1,25D3 for 24 h and followed by the combination of GC (gemcitabine dose range: 1.56–100 nM) for 48 h. The ratio of gemcitabine to cisplatin was fixed at 1:100. Cell growth was assessed by MTT assay. Drug interactions were quantitated by median-dose effect analysis, and combination index (CI) values were derived using CalcuSyn software (Biosoft, Ferguson, MO). CI values of <1,=1, and >1 indicate synergism, additive, and antagonism between the drugs, respectively.

In vitro Clonogenic assay

T24 cells were pretreated with ETOH or 1 μM 1,25D3 for 24 h followed by 1.25 nM gemcitabine and 0.1μg/ml cisplatin and subjected to the in vitro clonogenic assays as described.20

siRNA transfection

Synthetic small interfering RNA (siRNA) siGENOME SMARTpool siRNAs (4 individual siRNA pooled together) specific for p73, siCONTROL non-specific siRNA (siRNA-NS), and DharmaFECT1 transfection reagent were from Dharmacon (Lafayette, CO). T24 cells were transfected with 50 nM siRNA-NS or siRNA against p73 for 48 h using DharmaFECT1 transfection reagent following the manufacturer’s protocol.

In vivo excision clonogenic assay

The in vivo effect of 1,25D3 on GC-mediated antitumor activity was evaluated using a human T24 xenograft tumor model. Adult homozygous nude mice (Charles River Laboratories) were s.c. injected with 4 ×106 T24 cells, log-growth phase in 0.1 ml of Matrigel + HBSS (1:1), in the right rear flank. At day 8–9 post implantation, when the tumors were palpable (~ 6.5 × 5 mm), the mice (4/group) were treated with saline, 1,25D3 (0.625 μg/mouse/d, on day 1, 2 and 3), gemcitabine (6 mg/mouse/d, qd × 1, on day 2), cisplatin (0.12 mg/mouse/d, qd × 1, on day 3); or three drug combination (1,25D3 + GC). Twenty-four hours after the last treatment, tumors were harvested and in vivo excision clonogenic assay was performed as described.20

Tumor regrowth delay

The T24 xenograft tumor model was used and the mice were treated as described in the in vivo excision clonogenic assay. Tumor measurements were taken as described.21

Statistical analyses

Statistical significances between groups were determined by two-tailed student’s t-test.

RESULTS

Vitamin D receptor is expressed in human bladder cancer cells

1,25D3 exerts most of its activities through the vitamin D receptor (VDR), which is expressed in a wide range of cells and tissues. VDR protein expression in human bladder cancer cell lines T24 and UMUC3 were examined by immunoblot analysis. T24 cells did not express detectable VDR and 1,25D3 induced VDR expression in a dose dependent manner (Fig. 1). UMUC3 cells expressed a higher level of endogenous VDR, which was markedly induced by 1,25D3 (Fig. 1).

FIGURE 1.

FIGURE 1

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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.

1,25D3 promotes GC-induced apoptosis

To examine whether 1,25D3 promotes GC-induced apoptosis in bladder cancer cells, T24 or UMUC3 cells were treated with vehicle control ETOH, 1,25D3, gemcitabine, cisplatin, the combination of GC, or pretreated with 1,25D3 for 24 h followed by GC for 48 h and apoptosis was assessed by annexin V staining. 1,25D3 alone did not induce apoptosis in T24 (Fig. 2a) or UMUC3 cells (Fig. 2b). Gemcitabine or cisplatin alone induced apoptosis in T24 cells (Fig. 2a) and to a lesser extent in UMUC3 cells (Fig. 2b). Apoptosis was slightly increased by the combination of GC (Fig. 2a and b), which was further increased significantly by the pretreatment with 1,25D3 (Fig. 2a and b). To examine the involvement of caspases in 1,25D3 potentiation of GC-induced apoptosis, immunoblot analysis and substrate-based caspase activity assay were employed. 1,25D3 alone did not induce the cleavage of caspases- 8, 10, 9, 3 and poly (ADP-ribose) polymerase (PARP) as compared with control in T24 cells (Fig. 3a). Gemcitabine or cisplatin alone induced the cleavage of caspases- 8, 9 and PARP, which was not further enhanced by the combination treatments (Fig. 3a). Increased cleavage of caspase 3 was observed with the combination treatments (Fig. 3a). Similar results were observed in UMUC3 cells, but to a lesser extent (Fig. 3a). The more quantitative caspase assays revealed that GC treatment resulted in increased activities of caspases 8, 9 and 3, which was significantly (P < .05) enhanced by the pretreatment with 1,25D3 in T24 cells (Fig. 3b–d). Together, these results indicate that 1,25D3 enhances GC-induced apoptosis in bladder cancer cells, and this enhancement involves the activation of caspases 8, 9 and 3.

FIGURE 2.

FIGURE 2

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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.

FIGURE 3

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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.

1,25D3 promotes GC-mediated growth inhibition

Since 1,25D3 potentiated GC-induced apoptosis in bladder cancer cells especially T24 cells, we next investigated their effects on growth inhibition using MTT assay. T24 cells were treated with a series of doses of 1,25D3 for 24 h following by varying doses of GC, at a fixed ratio of 1:100, for 48 h. MTT results showed that pretreatment with 1,25D3 resulted in enhanced growth inhibition compared to GC combination (Fig. 4a). Standard median-dose effect isobologram analysis 22 was used to evaluate the nature of the interaction between 1,25D3 and GC. The combination index (CI) plotted against a series of treatment groups (Fig. 4b) or fraction affected (Fig. 4c) showed that the interaction between 1,25D3 and GC was synergistic at certain doses (CI < 1). The more sensitive in vitro clonogenic assay revealed that 1,25D3 alone did not alter the clonogenic capacity of T24 cells (Fig. 4d). The GC combination markedly suppressed the surviving fraction (Fig. 4d); this effect was significantly enhanced by pretreatment with 1,25D3 (Fig. 4d). These data indicate that 1,25D3 enhances GC-mediated growth inhibition.

FIGURE 4.

FIGURE 4

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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.

1,25D3 and GC induce p73 accumulation

We have previously demonstrated that 1,25D3 induces p73 accumulation in squamous cell carcinoma (SCC) cells, which contributes to 1,25D3-potentiated cisplatin antitumor activity.23 To examine whether p73 plays a role in 1,25D3 enhanced growth inhibition by GC in bladder cancer cells, the protein levels of TAp73 (p73) were assessed by immunoblot analysis using a monoclonal antibody targeting TAp73 but not ΔNp73 nor p53. Compared with the control, 1,25D3, gemcitabine, or cisplatin alone markedly increased p73 level in T24 cells, which was further increased with the combination treatment (Fig. 5a). Likewise, p73 was increased in UMUC3 cells, but more modestly (Fig. 5a). To better understand the involvement of p73 in bladder cancer, p73 protein levels in primary human bladder cancer tissues and adjacent normal tissues were examined by immunoblot analysis. Lower levels of p73 were observed in tumor tissues compared to control normal adjacent tissues in 3 out of 4 pairs of samples (#1, #3, and #4) (Fig. 5b), suggesting that increasing p73 level may be beneficial in treating bladder cancer.

FIGURE 5.

FIGURE 5

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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.

p73 contributes to 1,25D3 and GC-mediated growth inhibition

To investigate whether p73 contributes to 1,25D3-potentiated GC-induced growth inhibition, siRNA was used to knock down p73 expression as shown by immunoblot analysis (Fig. 5c). The following in vitro clonogenic assay showed that siRNA-p73 significantly (P < .01) enhanced the surviving fraction of T24 cells treated with 1,25D3, GC combination, or the pretreatment with 1,25D3 followed by GC compared with non- specific siRNA control (Fig. 5d). These results indicate that p73 plays a role in the growth inhibition mediated by 1,25D3 and GC.

1,25D3 enhances the antitumor activity of GC in vivo

Since 1,25D3 potentiated GC-mediated growth inhibition in T24 cells in vitro, we next investigated whether 1,25D3 had the same effects in vivo using the T24 xenograft model. As an indication of in vivo anti-tumor activity, in vivo excision clonogenic assay revealed that 1,25D3 alone or GC combination resulted in reduced surviving fraction (Fig. 6a). Pretreatment with 1,25D3 significantly suppressed the surviving fraction ascompared with 1,25D3 (P < .001) or the GC combination (P < .001) (Fig. 6a).

FIGURE 6.

FIGURE 6

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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.

To determine whether the results of in vivo excision clonogenic assay translate to antitumor effect in vivo, T24 tumor-bearing mice were treated with saline, 0.625 μg 1,25D3 daily for 3 d, 6 mg/mouse gemcitabine on day 2 and 0.12 mg/mouse cisplatin on day 3, or the combination of 1,25D3 and gemcitabine/cisplatin. 1,25D3 alone or GC treatment resulted in tumor regrowth delay compared with the saline control (Fig. 6b). Notably, the combination treatment with 1,25D3 and GC significantly (P < .05) further suppressed tumor growth (Fig. 6b). Mice treated with saline were sacrificed early due to tumor burden. These results indicate that 1,25D3 enhances in vivo antitumor activity of GC in the T24 xenograft model.

DISCUSSION

Metastatic bladder cancer has often been treated with standard multidrug chemotherapy consisting of methotrexate, vinblastine, doxorubicin and cisplatin (MVAC).3 However, MVAC regimen is highly toxic and better regimens were sought. GC combination is an effective regimen and has response rates, disease progression rates and overall survival rates comparable to MVAC.24 Importantly, the GC regimen is much safer and better tolerated.24 As a result, the GC regimen is widely accepted as a standard of care for patients with advanced bladder cancer. However, the response rates to these regimens are only around 50%.24 Inherent or acquired drug resistance is frequently observed and remains a major clinical problem. Tumor resistance to gemcitabine or cisplatin occurs through many mechanisms, and among which the inactivation of apoptotic pathways is a common and important mechanism.25, 26 Therefore, the development of new treatment options to increase chemosensitivity is needed.

1,25D3 has a broad spectrum of antitumor effects.12 It also additively or synergistically enhances antitumor activities of chemotherapeutic agents such as cisplatin and paclitaxel.12 Several mechanisms have been proposed for the enhanced antitumor effects. 1,25D3 pretreatment followed by cisplatin increases the expression of mitogen-activated protein kinase kinase kinase (MEKK-1) and caspase 3 activation, which may contributes to increased antitumor activity of cisplatin in SCC cells.27 1,25D3 sensitizes breast cancer cells to doxorubicin through the reduction of expression and activity of cytoplasmic antioxidant enzyme Cu/Zn superoxide dismutase, which increases the oxidative damage by doxorubicin.28 1,25D3-mediated enhancement of the growth inhibitory effects of paclitaxel in associated with increased Bcl-2 phosphorylation in breast cancer cells 29 and decreased p21 in prostate cancer cells.21

1,25D3 signals primarily through its receptor, VDR, which is a ligand-dependent transcription factor. VDR expression is detected in bladder tumor tissues with more positive cells in advanced tumor.30 Higher VDR expression is associated with a higher rate of progression in pathological stage.31 In comparison, VDR expression is low in normal urothlium.30 In the current study, we showed that human bladder cancer cell lines T24 and UMUC3 both expressed VDR which was further induced by 1,25D3. These results show that bladder cancer cells are capable of responding to 1,25D3.

Our laboratory has demonstrated that the optimal treatment schedule for greater cytoxicity in the combination therapy is pretreatment with 1,25D3 for 24 h followed by chemotherapeutic agents such as cisplatin and paclitaxel.21, 27, 32 We investigated whether 1,25D3 induced apoptosis in bladder cancer cells and whether pretreatment with 1,25D3 promotes GC-induced apoptosis in these cells. Using annexin V staining, we showed that 1,25D3, as a single agent, did not induce apoptosis in T24 or UMUC3 cells. Pretreatment with 1,25D3 significantly enhanced apoptosis induced by the combination of GC in both cell lines. Supporting these observations, 1,25D3 increased the activation of caspases- 8, 9 and 3 induced by GC. Further, 1,25D3 promoted GC-mediated growth inhibition as assessed by the MTT assay and in vitro clonogenic assay. The nature of the interaction between 1,25D3 and GC was synergistic using standard median-dose effect isobologram analysis. In line with our observations, a previous study showed that IC50 of 1,25D3 in T24 and another bladder cancer cell line 253j was 40μM and low concentrations of 1,25D3, as used in our current study, did not induce substantial cytotoxicity.33

As a tumor suppressor, p53 is frequently mutated in bladder cancers and p53 mutations are linked to tumor progression and poor prognosis.34 In this case, the status of p53 family member p73 may be important in tumor development and chemosensitivity.35 p73 is induced by various chemotherapeutic drugs and abrogation of p73 function results in chemoresistance.35 p53 family members also function as a network to modulate chemosensitivity. Mutant p53 has inhibitory effects on p73 and knockdown of mutant p53 promotes chemosensitivity.35 A p53 polymorphism at codon 72 (72R or 72P) in head and neck cancer is associated with the clinical response to chemotherapy, and the p53 mutants that inhibit p73 function in vitro are associated with more resistance to chemotherapy.36 A p53-derived peptide activates p73 and induces p73-dependent apoptosis in vitro and tumor regression in vivo.37 Using immunoblot analysis, we showed that p73 expression was lower in bladder tumor in three out of four pairs of adjacent normal tissue and tumor tissue. In line with our finding, low p73α expression was detected in 68% of primary human bladder tumors.11 The loss of p73α expression is also associated with tumor stage.11 We previously identified p73 as a target of 1,25D3, the level of which increased upon 1,25D3 treatment.23 Here we show that 1,25D3 also induced the accumulation of p73 protein in bladder cancer cell lines, especially in T24 cells. Knockdown of p73 by siRNA rescued the clonogenic ability of T24 cells treated with 1,25D3 alone, GC or the combination of 1,25D3 and GC, suggesting that p73 plays a role in growth inhibition mediated by these agents.

Further, 1,25D3 potentiated GC-mediated antitumor activity in vivo. T24 tumor cell clonogenic ability was suppressed by 1,25D3 alone or GC, and further suppressed by the combination of 1,25D3 and GC, as assessed by in vivo excision clonogenic assay. Employing the T24 human bladder xenograft model, we showed that the combination of 1,25D3 and GC also resulted in greater tumor regression compared with 1,25D3 alone or GC treatment. It is intriguing that 1,25D3 treatment alone did not induce apoptosis or growth inhibition in T24 cells in vitro; however, it exhibited antitumor effect in vivo. These observations suggest 1,25D3 may have an impact on tumor microenvironment. One potential mechanism for these observations is the effect of 1,25D3 on angiogenesis. 1,25D3 has been reported to inhibit angiogenesis in a variety of angiogenesis models.12

1,25D3 has been utilized in a number of clinical studies. The early clinical trials of 1,25D3 were conducted in patients with leukemia and myelodysplasia. Despite the pro-differentiation effect on leukemia blast cells in vitro, the results in patients were disappointing due to the development of dose-limiting hypercalcemia.38, 39 More recently, multiple clinical trials have demonstrated that sufficient doses of 1,25D3 to achieve exposure similar to those seen in the in vitro models can be safely administered by high dose intermittent regimen.40–43 Our group has conducted several phase I trials of 1,25D3 in combination with cytotoxic drug, including 1,25D3 + paclitaxel and 1,25D3 + carboplatin.44 Dose-limiting toxicity was not encountered. These clinical trials provide evidence and support for the continued study of 1,25D3 in combination with cytotoxic cancer chemotherapies.

In summary, this study reports that 1,25D3 enhances the antitumor activity of GC in vitro and in vivo, and p73 induction may contribute to this growth inhibition. The addition of 1,25D3 to the GC regimen may increase the chemosensitivity of bladder cancer and potentially a better response rate to the GC combination.

Acknowledgments

Grant support: This study was supported by NIH/NCI grants CA067267 and CA085142 to Dr. Candace S. Johnson, and CA095045 to Dr. Donald L. Trump. It was also supported, in part, by the NCI Cancer Center Support Grant to the Roswell Park Cancer Institute (CA016056).

We thank Ms. Rui-Xian Kong for her technical support.

Footnotes

Financial disclosure: There are no financial disclosures from any authors.

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