MART-10, a New Generation of Vitamin D Analog, Is More Potent than 1α,25-Dihydroxyvitamin D3 in Inhibiting Cell Proliferation and Inducing Apoptosis in ER+ MCF-7 Breast Cancer Cells

Hormone antagonist therapy for estrogen receptor positive (ER+) breast cancer patients post radical surgery and radiation therapy has a poor prognosis and also causes bone loss. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is a potent antitumor agent in pre-clinical studies, but caused hypercalcemia when its effective antitumor doses were used. Therefore, we investigated the effects of a less-calcemic 1α,25(OH)2D3 analog, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (MART-10), on ER+MCF-7 cells. We demonstrate that MART-10 is 500- to 1000-fold more potent than 1α,25(OH)2D3 in inhibiting cell growth in a dose- and time-dependent manner. MART-10 is also much more potent in arresting MCF-7cell cycle progression at G0/G1 phase as compared to 1α,25(OH)2D3, possibly mediated by a greater induction of p21 and p27 expression. Moreover, MART-10 is more active than 1α,25(OH)2D3 in causing cell apoptosis, likely through a higher BAX/Bcl expression ratio and the subsequent cytochrome C release from mitochondria to cytosol. Based on our in vitro findings, MART-10 could be a promising vitamin D analog for the potential treatment of breast cancer, for example, ER+ patients, to decrease the tumor relapse rate and the side effect on bone caused by antihormone regimens. Thus, further in vivo animal study is warranted.


Introduction
Breast cancer ranks first globally among the most commonly diagnosed and cancer-related deaths in women [1]. Over 1.38 million new breast cancer cases and 458,400 breast cancer-related deaths have been reported worldwide in 2008. Estrogen receptor (ER), which is present in nearly 70% of all breast cancer patients, plays a crucial role in the progression of breast cancer [2]. Thus, ER antagonists, tamoxifen and raloxifene, have been widely used to treat breast cancer and have contributed to a better prognosis for ER positive (ER+) breast cancer. However, only a 50% reduction in tumor relapse has been achieved by ER antagonist therapy [3]. Furthermore, the antagonists have serious side effects on bone [4], which highlights the necessity of seeking alternative treatments for ER+ breast cancer.
Vitamin D is well known as a modulator of calcium and bone metabolism. For the past three decades, abundant 2 Evidence-Based Complementary and Alternative Medicine evidence has been accumulated to indicate that the active form of vitamin D, 1α,25-dihydroxyvitamin D 3 , 1α,25(OH) 2 D 3 , or calcitriol, possesses many actions not associated with calcium and bone metabolism [5]. They include antiproliferation, antiangiogenesis, proapoptosis, prodifferentiation, and immune regulation in a cell-and tissue-specific manner [5][6][7][8][9].
1α,25(OH) 2 D 3 exerts its effects through binding to vitamin D receptor (VDR). The receptor is expressed in most human cancer cell lines and its growth can be inhibited by 1α,25(OH) 2 D 3 [10][11][12][13][14]. However, the clinical application of 1α,25(OH) 2 D 3 is hindered by its lethal hypercalcemic sideeffect after its systemic administration at a concentration sufficient to inhibit tumor cell growth [15]. To overcome this drawback, thousands of vitamin D analogs have been synthesized aiming to minimize its calcemic side effect while maintaining or even potentiating the antitumor activities [16,17].
For breast cancer, 1α,25(OH) 2 D 3 and its analogs, including EB1089, ILX 23-7533, and 22-oxa-1α,25(OH) 2 D 3 , have been shown to be effective in suppressing breast cancer cell growth in vitro and in vivo either alone or in combination with other drugs [18]. However, no significant benefit on survival has been observed in clinical trials [19,20].
MART-10 (19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D 3 ) [21] has been shown to be more active in VDR transactivation [22]. Most importantly, MART-10 is far more potent in inhibiting liver and prostate cancer cell proliferation [11,22,23] and prostate cancer cell invasion [24], and it did not raise serum calcium in vivo in an animal model [24]. These findings suggest that MART-10 could be a good candidate for breast cancer treatment. We, therefore, study the antiproliferative and proapoptotic effects of MART-10 in ER+ MCF-7 breast cancer cells and the potential mechanisms involved.

Cell Proliferation Assay by Cell Number
Counting. Cell counting was conducted using a hemocytometer as previously described [11]. Cells were treated every two days and counted on day 7.

Cell Cycle Analysis by Flow Cytometry.
Flow cytometry for cell cycle analysis was performed using a FACSCalibur (BD Biosciences, San Jose, CA, USA) as described previously [11,25]. Briefly, after exposure for two days to indicated concentrations of 1α,25(OH) 2 D 3 , the cells were collected and fixed in ice-cold 75% ethanol at 20 • C overnight. The fixed cells were stained in propidium iodide (PI) buffer containing 100 mM sodium citrate, 0.1% Triton X-100, 0.2 mg/mL RNase, and 50 μg/mL PI at 4 • C for 1 h. Flow cytometry and cell cycle analysis were then performed using a FACSCalibur.

Apoptosis Analysis by Flow
Cytometry. MCF-7 cell apoptosis was analyzed using a flow cytometer with Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI) staining kit (Strong Biotech Corporation, Taiwan) to distinguish early apoptotic from necrotic cells as previously described [11,26]. Briefly, three days after the indicated concentrations of MART-10 or 1α,25(OH) 2 D 3 treatment, MCF-7 cell apoptosis was analyzed using a flow cytometer with Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI) staining. Apoptosis Detection Kit (Strong Biotech Corporation, Taiwan) was applied in the present study. Briefly, cells from each sample were suspended in a mixture of 2 μL Annexin V-FITC, 2 μL propidium iodide (PI), and 100 μL AnnexinV-FITC binding buffer and then incubated at room temperature for 15 min. According to the cell density, 0.4-0.8 mL binding buffer was added. The samples were analyzed using a flow cytometer FACS Calilbur (BD Biosciences). The cell population was separated into three groups, that is, live cells with a low level of fluorescence, apoptotic cells in the earlier period with green fluorescence (Annexin V positive), and necrotic and advanced stage apoptotic cells with both red and green fluorescence (Annexin V and PI positive).

Apoptosis
Analysis by TUNEL Assay. TUNEL assay was used to measure DNA fragmentation [27]. Briefly, cells were plated on autoclaved glass coverslips in six-well culture plates and treated with MART-10 or 1α,25(OH) 2 D 3 as indicated in the figure legends. Cellular DNA was stained with apoptosis detection kits (Millipore Billerica, MA, USA), and the assay was performed according to the recommendations from the manufacturer (Millipore Billerica).

Statistical
Analysis. The data from each group were compared by the student t-test. P-value < 0.05 was considered as

VDR Expression in MCF-7 Cells.
Since the genomic actions of 1α,25(OH) 2 D 3 are mediated through VDR, we first analyzed the expression of VDR in MCF-7 cells. The expression in MDA-MB-231 cells served as a negative control [28]. As demonstrated in Figure 1(a), VDR was highly expressed in MCF-7 cells (lanes 1, 3, and 5), whereas very little or no expression (lanes 2, 4, and 6) was found in MDA-MB-231 cells as previously reported [28].

Induction of Cell Cycle
Arrest at G 0 /G 1 Phase and the Cyclin Dependent Kinase (CDK) Inhibitors, p21 and p27, by MART-10 and 1α,25(OH) 2 D 3 in MCF-7 Cells. Since MART-10 and 1α,25(OH) 2 D 3 showed a significant inhibition in the growth of MCF-7 cells, we next conducted cell cycle analysis by flow cytometry to further understand the mechanisms mediating the inhibition. When MCF-7 cells were treated with 10 −8 , 10 −7 , and 10 −6 M 1α,25(OH) 2 D 3 for two days , the fraction of cells arrested at G 0 /G 1 phase increased by 5.81%, 13.34%, and 13.78%, respectively, whereas we observed an increase in cell arrest at G 0 /G 1 by 10.45%, 15.36%, and 19.93% in the presence of 10 −9 , 10 −8 , and 10 −7 M of MART-10, respectively, as compared to the controls ( Figure 2 and Table 1). It is clear that although either 1α,25(OH) 2 D 3 or MART-10 can significantly arrest MCF-7 cell cycle progression at G 0 /G 1 , MART-10 is much more potent than 1α,25(OH) 2 D 3 in this respect.

Effects of 1α,25(OH) 2 D 3 and MART-10 on MCF-7 Cell
Apoptosis and Apoptotic Protein Expression. To compare the apoptotic response induced by 1α,25(OH) 2 D 3 and MART-10 in MCF-7 cells, flow cytometry analysis coupled with staining cells with Annexin V (Annexin V-FITC) and PI was utilized [29] (Figure 4(A)). The quantitative numerical distribution of apoptotic cells from this analysis is presented in Table 2. 1α,25(OH) 2 D 3 at 10 −6 M induced MCF-7 cell apoptosis by increasing the late apoptotic cell population from 7.19% to 10.04%, while MART-10 at 10 −7 M was able to increase the late apoptosis cell population from 7.19% to 13.66%. The results are in agreement with those obtained by TUNEL assay (Figure 4(B), panels a, b, c, and d). The figure shows that 8.2% and 8% apoptotic cells were generated when MCF-7 cells were treated with 10 −6 M 1α,25(OH) 2 D 3 and 10 −7 M MART-10, respectively. Our results, therefore, indicate that MART-10 is about 10-fold more potent than 1α,25(OH) 2 D 3 in the apoptotic induction of MCF-7 cells.
Bax protein is a well-known proapoptotic protein, whereas Bcl-2 is a protein with antiapoptotic activity. Therefore, the higher Bax/Bcl-2 ratio has been used as an indicator for the expression and the subsequent release of cytochrome C into cytosol to trigger apoptosis. As shown in Figure 5(a), 10 −7 M MART-10 and 1α,25(OH) 2 D 3 increased the Bax/Bcl-2 ratio to 1.48 and 1.33 as compared to the controls, which is in agreement with a greater upregulation of cytochrome C expression over controls by MART-10 (2.35-fold) than by 1α,25(OH) 2 D 3 (1.64-fold) ( Figure 5(b)).

Discussion
The focus of this study was to investigate the antiproliferative and proapoptotic activities of MART-10 in the ER+ MCF-7 breast cancer cells which express high level of VDR ( Figure  1(a)). MART-10 is a new generation of 1α,25(OH) 2 D 3 analogs with a skeleton of "2α-(3-hydroxy)propyl group" and "19-nor" integrated into one molecule. Therefore, MART-10 possesses the combined characteristics of the noncalcemic nature of the 19-nor vitamin D compounds [30] as exemplified by the FDA-approved drug Zemplar or 19-nor-1α, 25(OH) 2 D 2 for the treatment of the secondary hyperparathyroidism, and the enhanced VDR binding property of 2α-(3-hydroxy)propyl compound [31,32]. Similar to Zemplar, MART-10 did not raise serum calcium in an in vivo animal model [23] and was more potent than 1α,25(OH) 2 D 3 in inducing VDR transactivation [22].
The effects of vitamin D are mainly mediated through the VDR-dependent genomic actions. Our results confirm the high level of VDR expression in MCF-7 cells and accordingly highly sensitive growth inhibitory responses to 1α,25(OH) 2 D 3 and MART-10 in a dose-and timedependent manner (Figures 1(b) and 1(c)). The low or no   . Actin was used as the loading control. The lower panel depicts the average radio of the dose-dependent p27 expression relative to actin expression from three independent experiments. Each value is a mean ± SD of three independent determinations. * P < 0.05, * * P < 0.001 versus control. expression of VDR in MDA-MB-231 cells (Figure 1(a)) is in agreement with the low antiproliferative activity caused by 1α,25(OH) 2 D 3 and MART-10 ( Figure 1(d)) in these VDRnull cells. Thus, the results clearly suggest that VDR plays a crucial role in the response of MCF-7 breast cancer cells to 1α,25(OH) 2 D 3 . . Along this line, Lopes et al. recently reported that VDR expression was high in benign breast lesions and diminished gradually in invasive breast cancer as the tumor progressed [33]. VDR expression has also been shown to be inversely related to breast cancer incidence [34]. Collectively, the findings suggest that dysregulation of VDR expression may contribute to the incidence and progression of breast cancer.
In addition, our data, showing a greater cell growth inhibition induced by MART-10 than by 1α25(OH) 2 D 3 on day 5 and day 7 (Figure 1(c)), suggest that the effective dose of MART-10 may be higher than that of 1α,25(OH) 2 D 3 , possibly because MART-10 is more bioavailable than 1α,25(OH) 2 D 3 due to the nature that MART-10 is more resistant to CYP24A1 degradation [22,23].
Our results show that although both 1α,25(OH) 2 D 3 and MART-10 are active in inhibiting the proliferation (Figures  1(b) and 1(c)), inducing the cell cycle arrest at G 0 /G 1 phase (Figure 2 and Table 1) and promoting the apoptosis of MCF-7 cells (Figure 4), MART-10 is far more potent than 1α,25(OH) 2 D 3 . The greater antiproliferative activity with MART-10 over 1α,25(OH) 2 D 3 may be explained at least in part by its greater stimulatory effects on the expression of two tumor suppressor genes, p21 and p27, which act as CDK inhibitors to inhibit the progression of cells into the S phase of the cell cycle (Figure 3). This finding is consistent with   several previous reports that showed that p21 and p27 were the genes targeted by 1α25(OH) 2 D 3 and, therefore, leading to the arrest of cell growth [11,35,36]. As demonstrated in Figure 4 and Table 2, MART-10 is also more active than 1α,25(OH) 2 D 3 in inducing apoptosis. Bax, a proapoptotic protein, works toward the initiation of apoptosis through promoting the release of cytochrome C from mitochondria into cytosol. Whereas, Bcl-2, an antiapoptotic protein, functions as a protector to stabilize the mitochondrial membrane from releasing cytochrome C [37]. Studying MCF-7 breast cancer cells, James et al. [38] and Simboki-Campbell et al. [39] reported that 1α,25(OH) 2 D 3 induced apoptosis by downregulating Bcl-2 protein expression, increased TRPM-2 (clusterin) mRNA expression, and increased DNA fragmentation after 1α,25(OH) 2 D 3 treatment. In our studies with MCF-7 cells, both 1α,25(OH) 2 D 3 and MART-10 increased the ratio of Bax/Bcl-2 and the subsequent release of cytochrome C (Figures 5(a) and 5(b)). However, MART-10 is more potent than 1α,25(OH) 2 D 3 .
The release of cytochrome C from mitochondria to cytoplasm is a trigger of apoptosis pathway, leading to the activation of intrinsic initiator caspase 9, which in turn activates executioner caspase 3 and caspase 7 [40]. To investigate whether caspases were involved in the vitamin D-induced apoptosis in MCF-7 cells, we performed western blotting to detect the expression of the active form of caspases 3, 7, 8, and 9 in the presence of 10 −7 M of 1α,25(OH) 2 D 3 or MART-10 for 5 days. We found that none of them was detected either with or without 1α,25(OH) 2 D 3 or MART-10 treatment (unpublished data). Our results are in agreement with the previously published observations by Narvaez and Welsh [41] and Jänicke et al. [42]. Collectively, we conclude that MART-10 and 1α,25(OH) 2 D 3 -mediated apoptosis in MCF-7 cells may be cytochrome C-related but caspases-independent, and MART-10 is more potent than 1α,25(OH) 2 D 3 in inducing apoptosis in MCF-7 cells.
Evidence-Based Complementary and Alternative Medicine 9

Conclusion
For premenopausal women with ER+ breast cancer, the choice for antihormone treatment is tamoxifen or raloxifene which binds to ER, whereas aromatase inhibitors are the major therapeutic antihormone agents for the postmenopausal women with ER+ breast cancer. The drawback of tamoxifen or raloxifene and aromatase inhibitors is that they globally attenuate estrogen receptor transactivation or estrogen synthesis. It may be undesirable for some tissues where estrogen is essential to maintain normal functions, such as bone which needs estrogen to stimulate bone formation. On the contrary, 1α,25(OH) 2 D 3 can selectively downregulate aromatase and ER-α expression in breast cancer cells [43,44]. Along this line, we have performed preliminary studies indicating that MART-10 is far more potent than 1α,25(OH) 2 D 3 in inhibiting ER-α expression in MCF-7 cells (unpublished observation). In conclusion, we show that MART-10 is much more potent than 1α,25(OH) 2 D 3 in inhibiting cell growth through arresting cell cycle progression at G 1 phase and inducing apoptosis. In addition, the more bioavailable character of MART-10 as compared to 1α,25(OH) 2 D 3 in MCF-7 cells and its noncalcemic nature in an animal model suggest that MART-10 has potential as a superior chemotherapeutic agent to replace or to be in combination with traditional antihormone therapy for the treatment of breast cancer, such as the ER+ breast cancer patients, to decrease the tumor recurrence and eliminate the side effect on bone caused by the antihormone treatments.