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Journal of Medical Colleges of PLA 23 (2008) 358–363

Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

Luo Lan1, Xu Xinghua1, Sun Wenjuan1*, Dong Liying2 1Department of Pharmacology, College of Pharmacy, Key Laboratory of Biochemical

Pharmacology of Chongqing, Chongqing Medical University, Chongqing 400016, China 2Department of Neurology, People’s Hospital of Qianxi County, Qianxi 064300,

Hebei Province, China

Received 24 June 2008; accepted 28 October 2008

AbstractObjective: To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma

MDA-MB-231 cells. Methods: Light microscopy was used to determine the effects of amlodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen (PCNA) and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results: Amlodipine concentration of 8.25 mol/L (1/2 of IC50) affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion: The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.

Keywords: amlodipine, apoptosis, human breast carcinoma MDA-MB-231 cells, Bcl-2, proliferating cell nuclear antigen

1. Introduction Intracellular Ca2+ is indispensable to the cell

growth. The intracellular Ca2+ concentration is controlled by Ca2+ entry pathways in the plasma

* Corresponding author.

Tel.: 86-23-68485070 E-mail address: swenjuan6@yahoo.com.cn (Sun W.)

membrane and Ca2+ release from internal Ca2+stores located in the sarco (endo) plasmic reticulum. Ca2+ channel blockers have been known for a long period of time for their effectiveness in the treatment of hypertension, angina, and ventricular tachyarrythmia due to their inhibitory effect on Ca2+ entry via an interaction with the 1 subunit of the voltage-dependent L-type Ca2+ channel on the plasma membrane. However, in

Luo Lan et al. / Journal of Medical Colleges of PLA 23 (2008) 358–363 359

addition to their blockade of L-type Ca2+ channels, interactions with targets other than L-type Ca2+ channels, will result in ancillary effects in both vascular and non-vascular tissues. Such as reversal of drug resistance to chemotherapy in tumor patients, protection of ischemic tissue and inhibition of invasion of human breast carcinoma cells MDA-MB-231 [1]. The antiproliferative effects of several kinds of Ca2+ channel blockers on vascular smooth muscle cells and a variety of neoplastic cell lines have been demonstrated [2–4].

Amlodipine, a dihydropyridine Ca2+ channel blocker, is reported to inhibit proliferation of some cell lines. Our previous study [5] was also demonstrated that amlodipine inhibits human breast carcinoma MDA-MB-231 cells proliferation the IC50 value was 16.50 mol/L and was both dose- and time-dependent. Yoshida et al [6] demonstrated that amlodipine inhibits the growth of A431 cells by blunting the thapsigargin- or cyclopiazonic acid-induced Ca2+ release from endoplasmic reticulum and the ensuing Ca2+ influx through Ca2+-permeable channels, and A431 cells lack classical voltage-activated Ca2+ channels found in nerves and muscles. Thus, these results suggest that amlodipine may act through a mechanism by interfering with the intracellular Ca2+ regulating system responsible for internal Ca2+ stores, but not L-type Ca2+ channels.

Although interfering the intracellular Ca2+ mobilization has been demonstrated to be related to the antiproliferative action of amlodipine, the precise mechanisms of the action of the drug triggered by the inhibition of proliferation in neoplastic cells is still unknown. Since the oncogenesis is believed to be the result of the imbalance of proliferation and apotosis, and many anticancer drugs exert their cytotoxic effect by activating of apoptosis, in this study, therefore we examined the mechanism underlying the antiproliferative action of amlodipine by quantifying apoptosis rate (AR) with flow cytometry (FCM) and detecting the expressions of

PCNA and Bcl-2 protein with immunocyto- chemistry.

2. Material and methods

2.1. Reagents

Amlodipine (Lvdi pharmaceutical company, Shanghai) were dissolved in dimethylsufoxid (DMSO) to prepare a solution of 100 mmol/L. Annexin V-FITC cell apoptosis detection kit was purchased from Kaiji Biotechnology Company, Nanjing, and PCNA and Bcl-2 detection kit was from Zhongshan biotechnology company, Beijing.

2.2. Tumor cell lines

The human breast carcinoma MDA-MB-231 cells were kindly supplied by Professor Tang Weixue (Department of Pathophysiology, Chong- qing Medical University). The cells were cultured in RPMI 1640 supplement with 10% fetal bovine serum and 2 mmol/L glutamine at 37 under 5% CO2 in humidified atomosphere. Exponentially growing cells were used throuhout all the experiment at a density of 5×104/ml–5×106/ml.The cells were treated with amlodipine at a concentration of 8.25 mol/L (1/2 of IC50). After amlodipine treatment, cells were harvested by centrifugation at 800×g for 5 min, washed with cold RPMI 1640, and recentrifuged again at 800×g for 5 min. Cell number and viability were estimated by trypanblue in a hemocytometer.

2.3. Cell morphology

Human breast carcinoma MDA-MB-231 cells were treated by amlodipine with a final concentration of 8.25 mol/L (1/2 of IC50) and dissolvents for 48 h. Morphologic analysis was done by using an Olympus inverted transmitted light microscope at 20× magnification, containing HAL 6 V 25 W illumination. Imaging was done by using a Canon digital camera.

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2.4. Flow cytometric analysis of apoptosis

Annexin-V-FITC/PI double staining flow analysis of cytometry was used to evaluate cell apoptosis by using an Annein-V-FITC detection kit according to the manufacturer’s instructions. Human breast carcinoma MDA-MB-231 cells (5×106/ml) were plated in a 100-ml culture flask in RPMI 1640 containing 10% fetal bovine serum and cultured for 24 h. When cells in a good viability, amlodipine at a final concentration of 8.25 mol/L (1/2 of IC50) or solvents were added and incubated for 2 h. After cell suspension was made by trypsinization (0.25% of trypsin), the cells were washed and suspended with PBS-Buffer, reacting for 10 min in dark at room temperature(25±2 ) in a solution supplemented with 500 l of binding buffer, 2 l of Annexin -FITC and 5 l of Propidine Iodide of PBS-Buffer and filtered through a 25-mm Filter Tip (NPE systems). Annexin-V-FITC/PI stained samples were run on a NPE QuantaTM flow analyzer fitted with a mercury arc lamp, a 450nm H-ion exciter, a 565DCXR splitter (long pass) and a 450AF 525 nm, 620 nm red and green fluor emission filter. A minimum of 10 000 nuclear signals was collected in list mode files. Expo32 MultiCOMP software was used to analyze the samples

2.5. Proliferating cell nuclear antigen (PCNA) and Bcl-2: immunocytochemistry and labeling

By using PCNA and Bcl-2 detection kithuman breast carcinoma MDA-MB-231 cells were plated at densities of 5×104 /slide, Chamber slides used, and allowed to attach overnight. Then the cells were treated with amlodipine at a final concentration of 8.25 mol/L (1/2 of IC50) or solvents and incubated for 48 hours. Prior to immunostaining, the cells were rinsed thrice for 5 min in 1×automation buffer. The cells were fixed consecutively with 70% methanol and 4% paraformaldehyde for 10min each. Anti-PCNA or

Anti-Bcl-2 (1:75 dilution) and goat antimouse IgM (1:400 dilution) were used as the primary and secondary antibodies respectively. A Super Sensitive Link-label IHC Detection System was used according to the manufacturer’s recommend- dations. The kit contained 3.3’-diamino-benzidine (DAB) solution as the chromogen for visualization of staining. The cells were counterstained using Mayer’s hematoxylin. Chromatosis of brown or buffy grains located in cell nucleus was positive expression. HSCORE scoring was introduced to determine the positive expression of PCNA or Bcl-2. Chromatosis nuclei were graded as followsno chromatosis, recorded as 0 score; amber, recorded as 1 score; buffy, recorded as 2 score; and brown, recorded as 3 score. Calculation of the scores according to the following equation: HSCORE= pi(i+1)(i=0, 1, 2, 3; pi indicates the ratio of i scores).

2.6. Statistical analysis

The data was represented in mean±SD, differences between the control and the treated groups were analyzed using analysis of variance (ANOVA); differences between the control and the treated groups were analyzed using nonparametric statistics, differences were considered statistically significant when P<0.05.

3. Results

3.1. Cell morphology of MDA-MB-231 cells 48h after amlodipine treatment

Control group of human breast carcinoma MDA-MB-231 cells appeared robust and were elongated (Fig. 1), and there was cellular crowding, suggestive of proliferation. After treatment with amlodipine of 8.25 mol/L (1/2 of IC50) of human breast carcinoma MDA-MB-231 cells were shrunken and fewer in number (Fig. 1), furthermore, the cells were no longer robust and elongated.

Luo Lan et al. / Journal of Medical Colleges of PLA 23 (2008) 358–363 361

3.2. Apoptosis of a MDA-MB-231 cells 2h after amlodipine treatment

At 2h, the percentage of apoptotic human breast carcinoma MDA-MB-231 cells were significantly higher (P<0.01) in amlodipine of 8.25

mol/L (1/2 of IC50) treatment group than in the control group and that in 5-FU 5.43 mol/L(1/2 of IC50) treatment group. See Fig. 2.

3.3. PCNA immunoexpression and labeling indices of MDA-MB-231 cells 48h after amlodipine treatment

At 48h, PCNA immunoexpression and labeling indices in human breast carcinoma MDA-MB-231 cells treated with amlodipine of 8.25 mol/L (1/2 of IC50), were decreased compared with control

cells (P<0.01, Fig. 3).

3.4. Bcl-2 immunoexpression and labeling indices of MDA-MB-231 cells 48h after amlodipine treatment

At 48h, Bcl-2 immunoexpression and labeling indices in human breast carcinoma MDA-MB-231 cells treated with amlodipine of 8.25 mol/L (1/2 of IC50), were decreased compared with control cells (P<0.05, Fig. 4).

4. Discussion

Yoshida et al [6] demonstrated that that amlodipine evoked a transient Ca2+ release from thapsigargin-sensitive internal Ca2+ stores in A431 cells. On the other hand, nicardipine and nimodipine, despite having an inhibitory effect on

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Fig. 2. Apoptotic ratio of human breast carcinoma MDA-MB-231 cells in the control group (A), 5-Fu (5.43 mol/L) treatment group (B), amlodipine (8.25 mol/L) treatment group (C), and their statistical analysis (D). aP<0.01 vs control group; bP <0.01 vs 5-FU group.

Fig. 1. Human breast carcinoma MDA-MB-231 cells in the control group (A) and amlodipine (8.25 mol/L) treatment group under an inverted transmitted light microscope (×200).

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362 Luo Lan et al. / Journal of Medical Colleges of PLA 23 (2008) 276–282

A431 cell growth, did not evoke such an effect. Soamlodipine may be exhibit an another way from others calcium antagonists in the effect on antiproliferation. Because the increase of Intranuclear Ca2+ concentration can not only initiate gene expression and cell cycle progression, but also activate degradative processes in programed cell death or apoptosis. Prolonged high [Ca2+] activates nucleases that cleave DNA and degrade cell chromatin. Ca2+ promotes DNA digestion by direct stimulation of endonucleases, or indirectly by its activation of Ca2+-dependent proteases phosphatases and phospholipases result- ing in a loss of chromatin structural integrity [7]. Now that amlodipine evoked a transient Ca2+ release from thapsigargin-sensitive internal Ca2+ stores, this may cause Intranuclear Ca2+ increase, resulting in apoptosis. So we designed this study to evaluate the effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells.

This study found that human breast carcinoma

MDA-MB-231 cells treated with a concentration of 8.25 mol/L (1/2 of IC50) of amlodipine was hypoplasia, which implied that amlodipine exhibited an antiproliferation effect on human breast carcinoma MDA-MB-231 cells, and these conclusion were consistent with our previous study by MTT [5]. Simultaneously, the expressions of Bcl-2 and PCNA were decreased and significant in apoptosis.

It is now widely accepted that apoptosis is a gene-directed process and can be seen, alongside more familiar gene-directed processes such as differentiation, as a part of the repertoire available to the cell to respond to external and internal stimuli.

PCNA acts as a processivity factor for DNA polymerases and and is required for DNA replication [8] and nucleotide excision repair. PCNA appears mainly in the late G1 phase near the G1/S transition border of the cell cycle and reaches a maximum in the middle to late S phase.

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Fig. 3. PCNA expression of human breast carcinoma MDA-MB-231 cells in the control group (A) and amlodipine (8.25 mol/L) treatment group (B)(IHC×200), and their statistical analysis (C). aP<0.01 vs control group.

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A B CFig. 4. Bcl-2 expression of human breast carcinoma MDA-MB-231 cells in the control group (A) and amlodipine (8.25

mol/L) treatment group (B)(IHC×200), and their statistical analysis (C). aP<0.01 vs control group.

Luo Lan et al. / Journal of Medical Colleges of PLA 23 (2008) 358–363 363

During DNA replication, PCNA is localized at the site of DNA synthesis and can be detected by immunostaining of S-phase nuclei. It has been reported that ultraviolet irradiation of human amnion cells under conditions that induced nucleotide excision DNA repair synthesis caused a significant change in the nuclear levels of PCNA staining in non-S-phase cells. This indicates that PCNA might be involved in DNA repair synthesis as well as in DNA replication.

The bcl-2 gene, which is located at chromosome 18q21, was first identified in studies of t(14:18) chromosome translation in B-cell leukemia and follicular lymphoma. The bcl-2 gene is reported to promote cell survival, development, maturation, and differentiation and to act to protect against programmed cell death. That is morpho- logically equivalent to apoptosis. Bcl-2 oncoprotein specifically suppresses apoptosis in a range of cell types in vitro studies and indicated that this molecule indeed control the molecular process of apoptosis. Bcl-2 protein is present in the long-living self-renewing populations of stem cells that line the basement membranes of several complex epithelia, which are believed to die due to programed cell death. These results suggest that decreased expression of PCNA and Bcl-2 expression might be associated cells apoptosis.

Here we have reported evidence that the decreased expression of Bcl-2 and PCNA after treated with amlodipine are related to the lack of prevention from apoptosis, we conclude that antiproliferation of amlodipine on human breast carcinoma MDA-MB-231 cells involves apoptosis. Up to now, several genes with Bcl-2 related sequences have been identified. Bcl-2 suppresses apoptosis in several systems and coexpression of Bax opposes this effect, to allow apoptosis to proceed under appropriate condition. Bax forms heterodimers with Bcl-2, and the ratio of Bax to Bcl-2 could be critical in determing the fate of the cell in response to stimuli that can induce apoptosis.

In conclusion, amlodipine exhibits an antiproliferation on human breast carcinoma MDA-MB-231 cells. This effects is involved in apoptosis. As the intracellular mechanism of apoptosis is not yet fully determined, further investigation will be needed.

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(Editor Guo Jianxiu)