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. 2013 Apr 2;12(8):1316–1325. doi: 10.4161/cc.24445

Evaluation of the potential therapeutic role of a new generation of vitamin D analog, MART-10, in human pancreatic cancer cells in vitro and in vivo

Kun-Chun Chiang 1,2, Chun-Nan Yeh 3, Jun-Te Hsu 3, Ta-sen Yeh 3, Yi-yin Jan 3, Chun-Te Wu 4, Huang-Yang Chen 1, Shyh-Chuan Jwo 1, Masashi Takano 5, Atsushi Kittaka 5, Horng-Heng Juang 6,*, Tai C Chen 7,*
PMCID: PMC3674095  PMID: 23549173

Abstract

Pancreatic cancer is a lethal disease with no known effective chemotherapy and radiotherapy, and most patients are diagnosed in the late stage, making them unsuitable for surgery. Therefore, new therapeutic strategies are urgently needed. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is known to possess antitumor actions in many cancer cells in vitro and in vivo models. However, its clinical use is hampered by hypercalcemia. In this study, we investigated the effectiveness and safety of a new generation, less calcemic analog of 1α,25(OH)2D3, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10), in BxPC-3 human pancreatic carcinoma cells in vitro and in vivo. We demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)2D3 in inhibiting BxPC-3 cell proliferation in a time- and dose-dependent manner, accompanied by a greater upregulation of cyclin-dependent kinase inhibitors p21 and p27 and a greater downregulation of cyclin D3 and cyclin-dependent kinases 4 and 5, leading to a greater increase in the fraction of cells in G0/G1 phase. No induction of apoptosis and no effect on Cdc25 phosphatases A and C were observed in the presence of either MART-10 or 1α,25(OH)2D3. In a xenograft mouse model, treatment with 0.3 µg/kg body weight of MART-10 twice/week for 3 weeks caused a greater suppression of BxPC-3 tumor growth than the same dose of 1α,25(OH)2D3 without inducing hypercalcemia and weight loss. In conclusion, MART-10 is a promising agent against pancreatic cancer growth. Further clinical trial is warranted.

Keywords: cell cycle, pancreatic cancer, vitamin D analog, BxPC-3, xenograft, chemotherapy, MART-10

Introduction

Pancreatic adenocarcinoma (PCA) is one of the most lethal human malignancies with the lowest five-year survival rate of just 5%1,2 and an almost 1:1 ratio of incidence to mortality. An estimate of 44,000 new cases will be diagnosed with PCA, and more than 37,000 will die from this disease in 2013 in the US.1 Because of the lack of early detection methods, high probability of metastasis and resistance to available chemotherapy,3,4 PCA is the only one of the current top-five cancer killers, for which both the incidence and mortality rates have not decreased in the past 10 y, and with a likelihood of moving from the fourth to the second leading cancer killer in this country as early as 2015.1 Currently, the only effective therapy is radical surgery to remove the tumors. However, this disease is usually diagnosed in the late stage,2 with 40% having distant metastases and 40% with locally advanced PCA,5,6 excluding most patients from being the good candidates for surgery. Moreover, even after resection, the overall 5-y survival rate only improves to about 10–29%.3,7,8 Thus, under such bleak conditions, seeking new strategies against PCA should be an urgent priority.

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3 or calcitriol], has been shown to regulate a variety of biological actions through its interaction with vitamin D receptor (VDR) in many cell types and tissues in a cell- and tissue-specific manner, including antiproliferation, antiangiogenesis, anti-inflammation, pro-apoptosis, pro-differentiation and immuneregulation.9-12 As many as 638 genes may be regulated by 1α,25(OH)2D3,13 and more than several dozens of human cancer cell lines, including prostate, breast, lung, liver, colon and pancreatic cancer cells, have been found to express VDR, and their growth can be inhibited by 1α,25(OH)2D3.14-19 However, the clinical application of 1α,25(OH)2D3 is impeded by its lethal side effect of hypercalcemia.20 Thus, vitamin D analogs with minimized calcemic effect and enhanced antitumor activity have been synthesized.19,21,22 Several of them, including seocalcitol (EB1089), 22-oxa-1,25-dihydroxyvitamin D3 (OCT) and 19-nor-1α,25-dihydroxyvitamin D2 (paricalcitol), have been studied and demonstrated to exert antitumor activities through promoting cell cycle arrest, apoptosis induction and cellular differentiation in pancreatic cancer cells in vitro23-26 and in the xenograft animal models.27,28 Among the analogs, only EB1089 has been studied in a phase II trial in patients with inoperable pancreatic cancer.29 Unfortunately, no significant improvement in survival was observed in this trial.

MART-10 or 19 nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3,30 a new generation of 1α,25(OH)2D3 analog, has been shown to induce at least 3-fold higher VDR transactivation and 100- to 1,000-folds greater inhibition in liver, prostate and breast cancer cell proliferation in vitro.14,31-33 The analog is also 500-fold more resistant to CYP24A1 degradation than 1α,25(OH)2D3.31 In addition, MART-10 did not raise serum calcium in an in vivo animal model.33 In the current study, we investigated the utility of MART-10 to inhibit the growth of BxPC-3, a pancreatic cancer cell line highly responsive to the growth inhibitory effect of 1α,25(OH)2D324-26,28 in vitro and in vivo. In the current study, we demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)2D3 in repressing BxPC-3 cell growth in cultures, likely through cell cycle arrest at G0/G1, without inducing apoptosis. Our results also strongly suggest that the cell cycle arrest is mediated by the upregulation of p21 and p27 and downregulation of cyclin D3, cyclin-dependent kinase (CDK) 4 and CDK6. In the xenograft animal model, the administration of 0.3 µg/kg of MART-10 twice weekly significantly inhibited BxPC-3 tumor cell growth without inducing hypercalcemia and weight loss. This is the first animal study to show that MART-10 is active in an in vivo antitumor model without inducing systemic side effects. Given the dismal prognosis of PCA and the lack of effective treatment at the present time, our current study provides a basis for a further clinical trial to apply MART-10 either alone or in combination with other chemotherapeutic agent(s) as an alternative regimen to treat PCA.

Results

Expression of VDR in BxPC-3 cells

Since most of the vitamin D actions are mediated through its interaction with VDR to initiate gene transactivation, we first analyzed the expression of VDR by western blotting in BxPC-3 cells. As demonstrated in Figure 1A, VDR is highly expressed in this pancreatic cancer cell line. The expression in MDA-MB-231 and MCF-7 cells is served as a negative and a positive control, respectively.19,34

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Figure 1. The expression of VDR and the antiproliferative activity of 1α,25(OH)2D3 and MART-10 in BxPC-3 cells. (A) The expression of VDR in BxPC-3, MCF-7 and MDA-MB-231 cells as determined by western blot method. An aliquot containing 40 µg of protein from cell extracts prepared from BxPC-3, MCF-7 and MDA-MB-231 cells, respectively, was loaded to each lane. (B) The dose-dependent inhibition of BrdU incorporation into BxPC-3 cells induced by incubating with different doses of 1α,25(OH)2D3 or MART-10 for 20 h. Cells were plated at 5,000 cells per cm2 in 35-mm dishes. 1 d after plating, cells were treated with 1α,25(OH)2D3 (10−9 to 10−6 M) or MART-10 (10−10 to 10−7 M) for 20 h and then measured by BrdU assay as described in the Materials and Methods. Each value is a mean ± SD of three to five determinations. *p < 0.05, ** p < 0.001 vs. control. (C) The effects of 7 d incubation with repeated treatments with 1α,25(OH)2D3 and MART-10 on the growth of BxPC-3 cells. Cells were plated at 5,000 cells per cm2 in 35-mm dishes. After plating, cells were treated with 1α,25(OH)2D3 (10−10 to 10−6 M) or MART-10 (10−12 to 10−7 M) on day 2, 4 and 6 for a total of three times when media were changed. Cells were harvested 2 d after the final treatment. Cell number counting was performed using a hemocytometer as previously described.36,37 Results are presented as the % of control. Each value is a mean ± SD of three to five determinations. *p < 0.05, **p < 0.001 vs. control. (D). The time-dependent inhibitory effects of 1α,25(OH)2D3 and MART-10 on the growth of BxPC-3 cells. Cells were grown and treated with 10−8 M of 1α,25(OH)2D3 or MART-10 on day 2, 4 and 6 after plating for the group with one, two or three treatments, respectively. Cell number counting was conducted 2 d after the final dosing. Results are presented as the % of control. Each value is a mean ± SD of three to five determinations. *p < 0.05, **p < 0.001 vs. control.

Antiproliferative effect of MART-10 and 1α,25(OH)2D3 on BxPC-3 cells

To compare the antiproliferative activity of MART-10 against 1α,25(OH)2D3 in pancreatic cancer cells, we chose the BxPC-3 cell line. The reason is that this cell line expresses VDR (Fig. 1A) and has been shown to be highly responsive to 1α,25(OH)2D3-induced cell growth inhibition.24,25,28Figure 1B shows that after 20 h of treatment, 1α,25(OH)2D3 caused a 50% ± 2, 79% ± 3 and 76% ± 2 inhibition at a concentration of 10−8, 10−7 and 10−6 M, respectively, reaching plateau at 10–7 M, as determined by BrdU assay. On the other hand, treatment with MART-10 for 20 h at a concentration of 10−10, 10−9 and 10−8 M repressed BxPC-3 cell growth by 46% ± 2, 68% ± 3, 83% ± 2, respectively. No further inhibition (82% ± 2) was observed at 10−7 M of MART-10.

The BrdU data obtained by a single dose, short-term treatment were confirmed by a longer-term, cell number counting method with three repeated dosing with either 1α,25(OH)2D3 or MART-10. As shown in Figure 1C, after a 7 d treatment, BxPC-3 cell growth was inhibited in a dose-dependent manner by 0 ± 2, 9 ± 4, 43 ± 7, 80 ± 6 and 90 ± 5% in the presence of 10−10, 10−9, 10−8, 10−7 and 10−6 M of 1α,25(OH)2D3, or by 3 ± 4, 17 ± 3, 50 ± 6, 68 ± 8, 87 ± 6 and 88 ± 4% in the presence of 10−12, 10−11, 10−10, 10−9, 10−8 and 10−7 M of MART-10, respectively. No effect was detected with 10−12 M MART-10 or 10−10 M 1α,25(OH)2D3. The results (Fig. 1B and C), therefore, suggest that MART-10 is about 100-fold as potent as 1α,25(OH)2D3 in repressing BxPC-3 cell growth.

A time-dependent inhibitory effect by 1α,25(OH)2D3 or MART-10 at 10−8 M on BxPC-3 cell growth was also observed (Fig. 1D). 1α,25(OH)2D3 inhibited the pancreatic cancer cell growth by 10 ± 3, 27 ± 10 and 53 ± 3% on the 3rd, 5th and 7th day, whereas 21 ± 5, 60 ± 5 and 79 ± 3% growth inhibition by MART-10 was observed on the 3rd, 5th and 7th day, respectively, as determined by cell number counting. Taken together, both 1α,25(OH)2D3 and MART-10 inhibited BxPC-3 cell growth in cultures in a time- and dose-dependent manner, with MART-10 being much more potent than 1α,25(OH)2D3 in this respect.

Induction of cell cycle arrest at G0/G1phase by MART-10 and 1α,25(OH)2D3 in BxPC-3 cells

Since MART-10 and 1α,25(OH)2D3 showed significant inhibition in the growth of BxPC-3 cells, flow cytometry was conducted to analyze the cell cycle distribution of BxPC-3 cells after treatment. As shown in Figure 2A and B, BxPC-3 cells were treated with 1α,25(OH)2D3 or MART-10 at concentrations ranging from 10−6 to 10−8 or 10−7 to 10−9 M for 24 h, respectively. 1α,25(OH)2D3 at 10−8, 10−7 and 10−6 M increase the fraction of cells at G0/G1 phase from 36.59% (control) to 39.91%, 51.75% and 68.66%, an increase of 2.72%, 15.16% and 32.07%, respectively. On the other hand, an increase of 24.35% (from 36.59 to 60.94%) of the fraction of cells at G0/G1 phase was obtained with 10−9 M of MART-10. No significant increase was observed at the higher concentrations of MART-10; a 25.78% (from 36.59 to 62.37%) and 24.79% (from 36.59 to 61.38%) increase was obtained with 10−8 and 10−7 M of MART-10, respectively. Our results clearly indicate that either 1α,25(OH)2D3 or MART-10 can significantly arrest BxPC-3 cell cycle at G0/G1 phase, and MART-10 is at least 100-fold more potent than 1α,25(OH)2D3 in this respect.

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Figure 2. Flow cytometry analysis of cell cycle distribution for BxPC-3 cells treated with 1α,25(OH)2D3 or MART-10. Effects of 1α25(OH)2D3 and MART-10 on the relative distribution of BxPC-3 cells at G0/G1, S and G2/M phase. BxPC-3 cells were treated with 1α,25(OH)2D3 from 10−8 to 10−6 M and MART-10 from 10−9 to 10−7 M for 1 d before cell cycle analysis was performed with a flow cytometer. (A) A representative DNA histogram for ethanol-(control group), 1α,25(OH)2D3- or MART-10-treated BxPC-3 cells was shown. The total DNA content of cells (x-axis) was obtained by staining with propidium iodide. Cells were analyzed by flow cytometry. The percentage of cells in each cell cycle phase was determined with the program ModFit. The first, large peak represents population of cells (y-axis) in G0/G1 phase; the second, small peak shows population of cells in G2/M phase, and the gray area between both peaks represents cells in S phase. (B) The distribution of BxPC-3 cells as percentage in each cell cycle phase presented as a bar figure (left panel) and an actual number (right panel).

Evaluation of apoptotic induction by 1α,25(OH)2D3 or MART-10 in BxPC-3 cells

The pro-apoptotic action of 1α,25(OH)2D3 has been shown in many cancer cells and may contribute to the antiproliferative effect of 1α,25(OH)2D3.12,19 Therefore, to evaluate whether 1α,25(OH)2D3 and MART-10 induce apoptosis in BxPC-3 pancreatic cancer cells, flow cytometry analysis coupled with staining cells with Annexin V (Annexin V-FITC) and PI to distinguish the early apoptosis from the late apoptosis and necrosis35 were performed. As shown in Figure 3, after 2 d of treatment, the percentage of both PI- and Annexin V-positive cells was similar in the control and the treated groups, indicating no apoptotic induction by 1α,25 (OH)2D3 or MART-10 in BxPC-3 cells.

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Figure 3. Effects of 1α,25(OH)2D3 and MART-10 on BxPC-3 cell apoptosis analyzed by flow cytometry with Annexin V-FITC and propidium iodide (PI) staining. Annexin V-FITC in conjunction with PI staining was used to distinguish early apoptotic (Annexin V-FITC-positive, PI-negative, bottom right quadrant of each panel) from late apoptotic or necrotic cells (Annexin V-FITC-positive, PI-positive; top right quadrant of each panel). Fluorescence intensity for Annexin V-FITC is plotted on the x-axis, and PI is plotted on the y-axis as shown in the upper panel. The lower panel shows the percentage of apoptotic cell after 2 d of 10−6 M 1α,25(OH)2D3 or 10−7 M MART-10 treatment,

Evaluation of G1 arrest-related cycline dependent kinase (CDK) inhibitors, in BxPC-3 cells treated with 1α,25 (OH)2D3 or MART-10

Since MART-10 and 1α,25(OH)2D3 were able to arrest BxPC-3 cell cycle progression at G0/G1 phase, we next examined the expression of five cycline-dependent kinase inhibitors (CKIs) which are known to play a role in G0/G1 arrest in the presence of 1α,25(OH)2D3 or MART-10 by western blot analysis. Figure 4A shows that p27 expression increases 1.35 ± 0.27, 1.9 ± 0.35 and 2.3 ± 0.3 times over the control upon treatment with 10−8, 10−7 and 10−6 M of 1α,25(OH)2D3, respectively. MART-10 at 10−9, 10−8 and 10−7 M upregulated p27 expression 2.5 ± 0.31, 3.1 ± 0.29 and 3.3 ± 0.38 times over the control (Fig. 4A). Regarding p21 expression, 1α,25(OH)2D3 induced 1.4 ± 0.3, 1.5 ± 0.28 and 1.75 ± 0.15 times over the control group at 10−8, 10−7 and 10−6 M, respectively, whereas MART-10, at 10−9, 10−8 and 10−7 M, induced the expression 1.9 ± 0.23, 2 ± 0.18 and 2.1 ± 0.32 times over the control, respectively (Fig. 4A). We further analyzed the expression of other G1 arrest-related CKIs, p15, p18 and p19. No significant difference in their expression was noted either in the presence of 1α,25(OH)2D3 or MART-10 (Fig. 4B). Collectively, we conclude that 1α,25(OH)2D3 and MART-10 are both able to upregulate p21 and p27, leading to cell cycle arrest at G0/G1 in BxPC-3 cells, and MART-10 is far more potent than 1α,25(OH)2D3 in this respect.

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Figure 4. Western blot analysis of p21 and p27 expression and the expression of other G1 arrest-related proteins in BxPC-3 cells treated with 1α,25(OH)2D3 or MART-10. (A) Upper panel: A typical dose-dependent upregulation of p21 and p27 protein expression in response to 2 d of treatment with 1α,25(OH)2D3 (10−8 to 10−6 M) or MART-10 (10−9 to 10−7 M). Each lane was loaded with 30 µg of protein extracted from the control and treated cells. Actin was used as a loading control. Lower panel: The average expression ratio of p21 (right lower panel) and p27 (left lower panel) relative to actin from three independent experiments. Each value is a mean ± SD of three independent determinations. *p < 0.05, **p < 0.001 vs. control. (B) Upper panel: The expression of p15, p18 and p19 in response to the treatment with 10−6 M 1α,25(OH)2D3 (D) or 10−7 M MART-10 (M) for 2 d. C represents the control group. Each lane was loaded with 30 µg of protein extracts. Actin was used as a loading control. Lower panel: The average expression ratio of p15, p18 and p19 relative to actin from three independent experiments. Each value is a mean ± SD of three independent determinations. (C) Upperpanel: The expression of cyclinD3, CDK4 and CDK6 in response to the treatment with 10−6M 1α,25(OH)2D3 (D) or 10−7M MART-10 (M) for 2 d. C represents the control group. Each lane was loaded with 30 µg of protein extracts. Actin was used as a loading control. Lower panel: The average expression ratio of cyclinD3, CDK4 and CDK6 relative to actin from three independent experiments. Each value is a mean ± SD of three independent determinations.

Evaluation of other G1 arrest-related proteins in BxPC-3 cells treated with 1α,25 (OH)2D3 or MART-10

Besides CKIs, the progression of cell cycle through G1 is also regulated by many cyclins, cycline-dependent kinases (CDKs) and Cdc25 phosphatases for the essential phosphorylation or dephosphorylation of kinases involved in the signal transduction. Therefore, we next checked the expression of cyclin D3, CDK4, CDK6, Cdc25A and Cdc25C in BxPC-3 cells after treating the cells for 2 d with 10−6 M of 1α,25(OH)2D3 or 10−7M of MART-10. As shown in Figure 4C, 1α,25(OH)2D3 and MART-10 significantly inhibited the expression of cyclin D3, CDK4, CDK6. No influence on Cdc25A and Cdc25C expression was observed (data not shown).

Evaluation of the in vivo safety of MART-10 administration in nude mice

The safety of treating nude mice with 1α,25(OH)2D3 (0.3 µg/kg body weight) and MART-10 (0.15 and 0.3 µg/kg body weight) two times per week via IP injection for 3 wk was evaluated by checking serum calcium concentration and body weight of the nude mice weekly or twice weekly. As shown in Figure 5A and B, no significant changes in serum calcium concentration and body weight during the experiment period were observed, indicating the no or less-calcemic character of MART-10 and the safety of in vivo application of MART-10 under current regimen.

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Figure 5. Evaluation of the safety and the antitumor effect of systemically administered MART-10 and 1α,25(OH)2D3 on nude mice inoculated with BxPC-3 cancer cells . BxPC-3 cells (2.5 × 106) were injected subcutaneously into the back area of each nude mouse. Four weeks later, each group of mice were administered with either ethanol vehicle, 0.3 µg/kg 1α,25(OH)2D3, 0.15 µg/kg MART-10, or 0.3 µg/kg MART-10 through intraperitoneal (IP) injection two times per week for 3 wk. The body weight, serum calcium, tumor volume were measured as indicated during the experimental period. After 3 wk, the mice were sacrificed and the tumors were harvested, weighed and IHC staining was performed. D(0.3), M(0.15) and M(0.3) represent 1α,25(OH)2D3(0.3 µg/kg), MART-10 (0.15 µg/kg) and MART-10 (0.3 µg/kg) treated group, respectively. (A) The average body weight change (mean ± SD) in each group during the treatment period. (B) The average serum calcium concentration change (mean ± SD) in each group during the treatment period. (C) The appearance of tumor on the back of nude mice at the end of 3 wk of treatment with either vehicle control, 1α25(OH)2D3 or MART-10. (D) The average tumor volume change (mean ± SE) in each group during the treatment period. (E) The average final tumor weight (mean ± SD) in each group at the end of treatment period.

Evaluation of in vivo antitumor effect of 1α,25(OH)2D3 and MART-10 on BxPC-3 cells

Four weeks after BxPC-3 cells were inoculated into the back of nude mice, 1α,25(OH)2D3 (0.3 µg/kg body weight) or MART-10 (0.15 and 0.3 µg/kg body weight) was administered two times per week for 3 wk. MART-10 at a dose of 0.3 µg/kg body weight significantly attenuated the increase in tumor volume as compared with the other groups (Fig. 5C and D). MART-10 at a lower dose of 0.15 µg/kg body weight exhibited a similar effect to the animals treated with 0.3 µg/kg body weight of 1α,25(OH)2D3. At the end of the study, tumors were harvested and weighed (Fig. 5E). The figure shows that the group treated with 0.3 µg/kg body weight of MART-10 has the lowest tumor weight, which is significantly lower than the control group and the group treated with 1α,25(OH)2D3 at 0.3 µg/kg body weight. Thus, our results clearly demonstrate that MART-10 is effective and safe to inhibit BXPC-3 cell growth in vivo in this xenograft model.

Discussion

Pancreatic cancer is a deadly disease with 75% of patients dying within one year of being diagnosed and a 5-y survival rate of approximately 5%.4 Besides early detection of the tumor for radical surgery, there are very few effective options to treat this disease. Therefore, seeking a new strategy to treat advanced PCA is justified and urgently needed.

One promising approach came from the findings that the active form of vitamin D and many of its analogs have antitumor properties in cultured tumor cells and xenograft animal models. The potential role of using vitamin D compounds in treating pancreatic cancer has been explored. Kawa et al. first reported that 1α,25(OH)2D3 and its less calcemic analog, OCT, significantly inhibited the proliferation of several pancreatic cancer cell lines in culture with the greatest activity found in BxPC-3 cells, which also have the highest VDR content by Scatchard analysis.25 In the same study, they further investigated the antitumor activity of these two vitamin D compounds in vivo and reported that only OCT significantly suppressed the growth of BxPC-3 xenografts at 2 μg per kg body weight. Similarly, another less calcemic analog of 1α,25(OH)2D3, EB1089, was shown to inhibit GER human pancreatic carcinoma cells in cultures and in tumor xenografts in vivo.27 Based on the results obtained from animal and culture studies using pancreatic cancer cells and other types of cancer cells,36-44 a phase II trial of EB1089 in patients with advanced inoperable pancreatic cancer was launched to determine the individual maximum tolerated dose (MTD) for each study subject and the antitumor response. They reported that most patients tolerated well a dose of 10–15 µg per day in chronic administration. Unfortunately, the drug had no objective antitumor activity.29

In our study, we show for the first time the expression of VDR protein in BxPC3 pancreatic cancer cells by western blot analysis, which supports the previous reports by Scatchard analysis25 and northern blotting.45 Most importantly, we demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)2D3 in inhibiting BxPC-3 cell growth in vitro. In addition, this is the first study to demonstrate the antitumor effect of MART-10 in vivo (Fig. 5C–E) without inducing hypercalcemia (Fig. 5B) and other toxicity such as weight loss (Fig. 5A). Using the same dosage, 1α,25(OH)2D3 showed much less effect on tumor weight and volume as compared with MART-10. Although there are no published data comparing the antiproliferative activity of MART-10 against EB1089 in pancreatic cancer cells available, it is reasonable to assume that MART-10 is 10–100-fold more active than EB1089 in inhibiting pancreatic cancer cell proliferation in vitro. This assumption is based on the published results comparing the antiproliferative activity of EB1089 against 1α,25(OH)2D336,38,39,41 and MART-10 against 1α,25(OH)2D319 in MCF-7 cells. Therefore, we believe that MART-10 is likely 10–100-fold more potent than EB1089 in inhibiting pancreatic cancer cells in vitro and xenograft models in vivo.

There are several reasons that may explain the greater antitumor activity we have observed with MART-10 over 1α,25(OH)2D3 in pancreatic cancer cells. First, Hourai et al. previously reported that 2α-(ω-hydroxypropyl)alkylated vitamin D3 analogs exhibited higher VDR binding affinity than that of 1α,25(OH)2D3.46 X-ray crystallographic analysis of the VDR-[2α-(3-hydroxypropyl)-1α,25(OH)2D3] complex has clearly demonstrated that the terminal hydroxyl of its 2α-(3-hydroxypropyl) forms a direct hydrogen bonding with Arg 274 of the VDR ligand binding domain (LBD), replacing one of the water molecules within the LBD binding pocket to stabilize the complex, resulting in a 3-times higher binding affinity for VDR47,48 and gene transactivation activity31 by MART-10 than by 1α,25(OH)2D3. Second, MART-10 has a much weaker binding to vitamin D binding protein (DBP).31 This property may allow MART-10 to be taken up from the circulation by pancreatic cancer cells to exert its genomic effects more easily than 1α,25(OH)2D3. Third, a cell-free reconstituted assay system has demonstrated that comparing to 1α,25(OH)2D3, MART-10 is more resistant to the CYP24A1-dependent 24-hydroxylation, the first step to degrade and terminate the actions of MART-10 and 1α,25(OH)2D3.31 The more resistant nature of MART-10 can be explained by a longer lasting upregulation of CYP24A1 by this analog than by 1α,25(OH)2D3 in PC-3 cells31 and MCF-7 cells (unpublished observation), and a docking model analysis of MART-10 bound to a computer generated human CPY24A1.49

During the cell cycle progression, E2F-1 transcription factor is bound to hypophosphorylated retinoblastoma protein (RB)50 and, thus, it is inactive. In mid to late G1 phase, specific cyclin-bound CDKs phosphorylate RB and displace the E2F-1 transcriptional factor, which further activates the gene expressions essential for the cell to enter S phase.51 The activity of cyclin-CDK complex required for G1/S transition is regulated by various endogenous CDK inhibitors (CKIs). Among them, p21 and p27 are the two major CKIs responsible for G1/S transition. As shown in Figure 2, 1α,25(OH)2D3 or MART-10 significantly repressed the BxPC-3 cell growth through cell cycle arrest at G0/G1 phase. MART-10 is much more potent than 1α,25(OH)2D3 to upregulate p21 and p27 (Fig. 4A) and, thus, could repress cell cycle progression at G0/G1 phase to a greater extent than 1α,25(OH)2D3. These findings could be attributed to the higher binding affinity of MART-10 to VDR and the more bioavailable nature of MART-10 as discussed earlier. The results are consistent with previous reports showing that p21 and p27 were the genes targeted by 1α,25(OH)2D3 and, therefore, lead to the arrest of cell growth.14,19,52,53 Our finding is also in line with previous studies showing that 1α,25(OH)2D3 and its analog could repress pancreatic cancer cell growth via cell cycle arrest at G0/G1 and upregulation of p21 and p27, followed by the downregulation of cyclins and CDK.24,28 Although other CKIs, such as p15, p18 and p19, have been implicated in G1/S transition,54,55 none of them was found to be involved in the MART-10- or 1α,25(OH)2D3-mediated cell cycle arrest in BxPC-3 cells (Fig. 4B). Besides CKIs, some cyclins and CDKs associated with G1/S transition were also investigated in this study. As shown in Figure 4C, 1α,25(OH)2D3 at 10−6 M and MART-10 at 10−7 M significantly repress cyclin D3, CDK4 and CDK6 expression in BxPC-3 cells. Others such as Cdc25 families are also known to play an important role in cell cycle progression.56 Therefore, we checked Cdc25A and Cdc25C expression in BxPC-3 cells after 1α,25(OH)2D3 or MART-10 treatment and found no significant change in the expression of Cdc25A and Cdc25C as compared with the control (unpublished observation). Taken together, we conclude that both 1α,25(OH)2D3 and MART-10 can effectively inhibit BxPC-3 cell growth mediated by cell cycle arrest at G0/G1 phase with upregulation of p21 and p27 (Fig. 4A) and downregulation of cyclin D3, CDK4 and CDK6 (Fig. 4C), and MART-10 is clearly much more potent than 1α,25(OH)2D3. Although 1α,25(OH)2D3 has been shown to induce apoptosis in other cancer cells,12,19 no apoptotic induction was detected in BxPC-3 cells in the current study (Fig. 3).

Even with the rapid progress in cancer treatment, pancreatic cancer remains a foremost challenge for physicians. Advanced PCA patients still have a dismal prognosis, with overall survival less than 1 y. In a phase II clinical trial enrolling 25 advanced pancreatic cancer patients, a combination of oral 1α,25(OH)2D3 (0.5 µg/kg) and docetaxel significantly increased the period of time-to-progress of pancreatic cancer as compared with treatment with docetaxel alone.57 In our current in vivo study, we demonstrated that MART-10 is non-calcemic under an effective dose of 0.3 µg/kg body weight and is more potent than 1α,25(OH)2D3 in repressing pancreatic cancer tumor growth. Thus, clinical trials using MART-10 either alone or in combination with docetaxel or other chemotherapeutic agents in advanced PCA patients are warranted.

Materials and Methods

Vitamin D compounds

1α,25(OH)2D3 was purchased from Sigma. 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 (MART-10) was synthesized as previously described.30

Cell cultures

Human pancreatic cancer cell line BxPC-3 was obtained from ATCC. BxPC-3 cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS). Culture medium was changed three times per week.

Cell proliferation assays

(1) Cell number counting: Cell counting was conducted using a hemocytometer as previously described.58,59 Cells were treated every 2 d with 1α,25(OH)2D3 or MART-10 for a total of three times and counted 2 d after the last dosing.

(2) BrdU assay: The BrdU assay was performed using a BrdU ELISA kit [Roche Cell Proliferation ELISA, BrdU (colorimetric) #11647229001, Roche Diagnostics, GmbH]. Briefly, cells were plated in a 96-well plate, cultured to approximately 50% confluence and treated with the indicated concentrations of 1α,25(OH)2D3 or MART-10. Twenty hours later, cells were harvested and labeled according to the procedures described in the kit manual.

Western blot

The procedures for protein extraction, blocking and immunodetection were described previously.14,19 The primary antibodies used in this study were mouse monoclonal antibodies against VDR (D-6, Santa Cruz Biotechnology), P15 (Cell Signaling, #4822), P18 (Cell Signaling, #2896), P19 (BDPharMingen, 610530), p21 (Cell Signaling, #2947), p27 (Cell Signaling, #3698), Cdc 25A (Cell Signaling, #3652), Cdc 25C(Cell Signaling, #4688), cdk4 (Cell Signaling, #2906), cdk6 (Cell Signaling, #3136), cyclin D3 (Cell Signaling, #2936). The secondary antibodies were anti-rabbit (Jackson Immunoresearch, 111-035-003) or anti-mouse secondary antibodies (Zymed 81-6520) (1:5,000). The blots were detected using ECL reagents (Millipore, WBKLS0500). Membranes were detected by VersaDoc™ Imaging System (Bio-Rad) for analysis.

Cell cycle analysis with flow cytometry

1 d after exposing to indicated concentrations of 1α,25(OH)2D3 or MART-10, the cells were collected and fixed in ice-cold 75% ethanol at 4°C overnight. The fixed cells were stained as described previously.14,19 Flow cytometry and cell cycle analysis were then performed using a FACS Calilbur (BD Biosciences) as described previously.60

Apoptosis analysis by flow cytometry

2 d after MART-10 (10−7 M) or 1α,25(OH)2D3 (10−7 M) treatment, BxPC-3 cell apoptosis was analyzed using a flow cytometer with Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI) staining.14,19 Apoptosis Detection Kit (Strong Biotech Corporation) was applied in the present study. The cell population was analyzed as previously described.61

Animal studies

Animal studies were approved by the experimental animal ethics committee at Chang Gung Memorial Hospital, and conformed to the US National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (Publication No. 85–23, revised 1996). Young male BALB/C Nu/Nu mice, age 6-wk-old and average 20–30 g, were purchased from National Taiwan Animal Center. They were housed in cages of six in a group and fed with rat chow and water ad lib in the animal facility of Chang Gung Memorial Hospital with strict adherence to the guidelines of Laboratory Animal Safety Committee. A total of 24 adult male young male BALB/C Nu/Nu mice were used in each experiment. The animals were divided into one control group (n = 6) and three experimental groups (n = 6 per group). BxPC-3 cells (2.5 × 106) were injected subcutaneously into the back area of each nude mouse. Four weeks later, each group of mice were administered with either vehicle, 0.3 µg/kg 1α25(OH)2D3, 0.15 µg/kg MART-10 or 0.3 µg/kg MART-10 by intraperitoneal (IP) injection two times per week for 3 wk. The body weight, serum calcium and tumor volume were measured during the experimental period. Serum calcium was measured by calcium assay kit (#Z5030014, Biochain). Tumor volumes were calculated using the Equation 1/6πd3. After 3 wk, mice were sacrificed and tumors were harvested, weighed and IHC staining was performed. The experiment was repeated two more times.

Statistical methods

The data from each group were compared by the Student’s t-test pvalue < 0.05 was considered as a significant difference. Differences between experimental animals and controls were calculated using the Mann-Whitney U test. The Excel 2007 SPSS statistical software program for Windows (SPSS version 10.0) was employed to conduct the statistics.

Acknowledgments

This work was supported by Chang Gung Medical Research Program (CMRP) grants 280271G, 280272G and 280273G (Dr Kun-Chun Chiang), and in part by CTSA grant UL1-TR000157 from NIH.

Glossary

Abbreviations:

1α,25(OH)2D3

1α,25-dihydroxyvitamin D3

MART-10

19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3

PCA

pancreatic adenocarcinoma

VDR

vitamin D receptor

EB1089

seocalcitol

OCT

22-oxa-1,25-dihydroxyvitamin D3

paricalcitol

19-nor-1α,25-dihydroxyvitamin D2

CDK

cyclin dependent kinase

FBS

fetal bovine serum

PI

propidium iodide

IHC

immunohistochemical

IP

intraperitoneal

Footnotes

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