Effects of Bufalin and Cinobufagin on the Androgen Dependent and Androgen Independent Prostate Cancer Cell Lines
Ching-Han Yu, Hui-Wen Hsu, Pei-Chi Chen, and Paulus S. Wang.
Department of Physiology, School of Medicine, National Yang-Ming University, Taipei (11221), Taiwan, Republic of China.
Correspondence: Paulus S. Wang. Ph. D.
Department of Physiology
National Yang-Ming University
Shih-Pai, Taipei, R. O. C.
Tel: 886-2-28267082
Fax: 886-2-28264049
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Running Title: BF & CB Inhibits Proliferation of Prostate Cancer Cells
Key Words: Chan su, topoisomerase Ⅱ, Na+-K+-ATPase, prostate cancer, apoptosis
ABSTRACT
Purpose: It has been well known that the extracts of Chan-Su are cardiac glycosides, and were used as treatments for heart failure. Chan-Su has been known to induce cytostatic or oncolytic effects in several cancers, but few studies about Chan-Su on prostate cancer cells have been reported. This study was to evaluate the anti-proliferation effects and the underlying mechanisms of bufalin and cinobufagin, the bufadienolides extracts from Chan-Su, on the androgen-dependent and -independent prostate cancer cell lines.
Experimental Design: Cell proliferation of three prostate cancer cell lines, LNCaP, DU145 and PC3, was measured by MTT assay, and calculated for 50% inhibition concentration (IC50) and doubling time (tD). Both western blot and colorimetric assay were used to analyze the protein expressions and activities of caspases. Other protein expressions of apoptosis modulators, such as mitochondrial Bax, and cytosolic cytochrome c, were also analyzed by western blot.
Results: Bufalin and cinobufagin inhibited cell proliferation of all three prostate cancer cell lines in a dose-dependent manner. The IC50 of bufalin on prostate cancer cell lines was less than that of cinobufagin. Therefore, the cytotoxicity of bufalin was more serious than that of cinobufagin. In addition, bufalin and cinobufagin caused cell growth doubling time changes in three prostate cancer cell lines, which was greater than those in human glomerular cells (human mesangial cells, HMC). An increase of the active form of caspases expression was observed in the bufalin or cinobufagin-treated cells, while the caspase activities were also elevated. Protein expressions of upstream apoptosis modulators, Bax and cytochrome c, were increased.
Conclusion: These results suggest that bufalin and cinobufagin suppress the proliferation and cause cell apoptosis in androgen-dependent and -independent prostate cancer cell lines via an increase of the expressions of a sequence of apoptosis modulators and effectors including Bax, cytochrome c, and a cascade of caspases.
INTRODUCTION
Prostate cancer has the highest incidence, and is the second most common cause of cancer death of men in the United States (1). In addition, the incidence and mortality of prostate cancer also increase in Asia countries for the past decade. Traditionally, radiation therapy and surgery are curative treatments for prostate cancer in situ. Androgen ablation is the mainstay treatment for metastatic prostate cancer; however, it results in the recurrence of androgen-independent cancer cells in 80% of patients, which has the median survival for six to nine months (2). There are still no effective chemotherapy drugs for hormone-refractory prostate cancer. Therefore, it is important to investigate a novel and effectual treatment for hormone-insensitive prostate cancer.
Bufalin, one of the prominent components of Chan su extracts from the venom of Bufo bufo gargarizan, is reported as a Na+-K+-ATPase inhibitor which results in an elevation of intracellular calcium concentration (3). On the basis of this mechanism, bufalin may increase vasoconstriction and blood pressure, and be used as a treatment for heart failure in Chinese medicine. Disruption of intracellular calcium homeostasis induces apoptosis in diverse cell types (4). Our previous results have illustrated that bufalin, with digitalis-like activity, is able to induce the increase of intracellular calcium and cell apoptosis in prostate cancer cells, but the detail mechanism remains unclear (5). On the other hand, bufalin is also shown as a topoisomerase Ⅱ inhibitor. Topoisomerase Ⅱ is a nuclear enzyme that relaxes supercoiled DNA at the time of DNA replication. Topoisomerase Ⅱ inhibitors, such as etoposide and adriamycin, maintain the complex of topoisomerase Ⅱ and the 5'-cleaved ends of the DNA resulting in protein-linked DNA double strand breaks (6). Owing to the mechanism described above, bufalin may induce leukemia cell differentiation (7) and apoptosis (8). Furthermore, inhibition of solid tumor growth (9), and endothelial cell proliferation and angiogenesis caused by bufalin in vitro has also been reported (10). Since inhibitors of topoisomerase and Na+-K+-ATPase have been demonstrated to induce apoptosis in some cancer cell lines, it is interested to investigate the effects of bufalin and cinobufagin, another major component of Chan su, in androgen-dependet, LNCaP, and androgen-independent, DU145 and PC3, prostate cancer cell lines.
Inducing cell apoptosis has been the target mechanism for chemotherapy drugs to treat a variety of cancer. Caspase cascade is a well known key pathway in the apoptotic signal transduction. Caspases are normally present in the cell as zymogens (procaspases), and could be divided into two types of subfamilies: upstream initiator caspases (caspase 8 and 9), which are involved in regulatory events, and downstream effector caspases (caspase 3, 6, and 7), which are directly response for the change of cell morphological events (11). There are two major apoptotic pathways on the upstream of caspase cascade to date, which are either the intrinsic pathway (the mitochondria) or the extrinsic pathway (the cell surface receptors) (12). Because of the character of bufalin as an inhibitor of topoisomerase Ⅱ, it may induce DNA damage and activate the mitochondrial pathway to regulate cell apoptosis. Bcl-2 family may be divided into two functional subfamilies such as pro-apoptotic proteins (Bax and Bid) and anti-apoptotic proteins (Bcl-2 and Bcl-xL). Bcl-2 family members translocate to mitochondria and mediate the membrane potential to induce cytochrome c release. The cytosolic cytochrome c is further involved in the signal transduction of caspase activation and finally cause cell apoptosis.
The rationale to develop bufalin as a potential therapy for prostate cancer is based on its ability to increase intracellular calcium concentration and to activate mitochondria-mediated cell apoptosis in a variety of cancer cells. The principal objectives of the present study were to: (a) characterize the morphological transformation induced by bufalin or cinobufagin, as well as the IC50 (50% of inhibition concentration) of both treatments in three prostate cancer cell lines; (b) determine the alteration of the doubling time (tD) generated by bufalin or cinobufagin during the time of cell proliferation in three kinds of prostate cancer cells and a normal human cell (human mesangial cells, HMC) for comparing the toxicity effects in normal cells with that in cancer cells; (c) explore the mechanism of antiproliferative effects occasioned by both bufadienolides depended on the measurement of caspase activation and protein expressions of caspases as well as the upstream molecules, Bax and cytochrome c. Such investigations may, in part, illustrate the antitumor effects of bufalin and cinobufagin in prostate cancer, and help to introduce a new treatment in cancer patients.
MATERIALS AND METHODS
Cells and Culture Conditions.
Androgen dependent human prostate carcinoma cell line LNCaP was purchased from Culture Collection and Research Center (CCRC) of Food Industry Research and Development Institute (FIRDI), Taiwan, ROC. Androgen independent prostate cancer cell lines DU145 and PC3 were provided by Dr. C. R. Jan (Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Kaohsiung, Taiwan, ROC). The primary cultured human mesangial cells (HMC) were generously given by Dr. L. Y. Yang (Department of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan, ROC). Cell lines were maintained in RPMI 1640 (Gibco Laboratories, Buffalo, Grand Island, NY, USA)(LNCaP) or in Dulbecco’s Modified Eagle’s Media (DMEM, Gibco Laboratories, Buffalo, Grand Island, NY, USA)(DU145 and PC3) with 50 IU/ml potassium penicillin G (Sigma, St. Louis, MO, USA), 50 IU/ml streptomycin sulfate (Sigma, St. Louis, MO, USA) and 10% fetal calf serum (FCS, PAA, Pasching, Austria) as standard media in an atmosphere of 5 % CO2 at 37℃. HMC were cultured in RPMI 1640 media with the presence of 200 mM glutamine, 100 mM sodium-pyruvate, 10 mM nonessential amino acid, 100 IU/ml insulin, 4 mg/ml transferrin, 50 IU/ml potassium penicillin G, 50 IU/ml streptomycin sulfate, 250 μg/ml fungizone, and 10% FCS at 37℃ in an atmosphere of 5 % CO2.
Cell Morphology Microscopy.
Cancer cells grown in 10-cm dishes for 24 h (DU145 and PC3) or 48 h (LNCaP) were challenged with different concentration of drug or a drug-free medium (control condition). At certain time points, cultures were photographed with a phase contrast microscope (Nikon, Chiyoda-Ku, Tokyo, Japan).
Cell Proliferation Assay.
Cell proliferation was determined by the modified colorimetric 3-(4,5-dimethylthiazol-2-yle)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cells were dispensed at a density of 2000/well of 96-well microplates in 100 μl medium supplemented with 10% FBS. After cell attachment, the media were replaced by the new media containing different concentration of bufalin or cinobufagin (day 0). The MTT (Sigma, St. Louis, MO, USA) assay was performed on day 1, 2, 3, and 4 modified from Janssen, et al., 1996. Briefly, the media were supplanted by 50 ml MTT solution (1 mg/ml in RPMI 1640 or DMEM). After 4-h incubation, the MTT solution was displaced by 50 ml DMSO, and the plates were shaken for 1 min. The optical density of each plate was quantified for absorbance at a wavelength of 570 nm with a reference wavelength of 630 nm using a microplate reader (Dynatech Laboratories, Chantilly, VA, USA). Proliferation index of each day referred to the optical density of that day divided by the optical density of day 0. Each experimental condition was performed in three preparations and repeated by four times.
Proliferation indexes of different concentration at day 1 to day 4 were calculated and plotted by fitting to the sigmoidal logistic four parameter nonlinear regression formula (SigmaPlot, Jandel Scientific Software, San Rafael, CA, USA) (14). IC50 value of each day was determined from plot of percent of untreated control cell growth versus the logarithm of the drug concentration.
To find out the change of doubling time caused by bufalin or cinobufagin at the concentration of 0.1 mM in HMC and three prostate cancer cell lines, the proliferation index were plotted on the common log scale and fit by linear regression using the equation: y = log(ODt/OD0) = (log2/tD)t, where ODt and OD0 represent the optical density at the day t or that at the day 0, respectively; tD represents for doubling time. The doubling time was calculated by tD = Log2/S (days), where S is the slope of the regression line (15).
Caspase Activity Analysis.
Caspase activation was measured in cytosolic extracts using the peptide substrates, DEVD-pNA, IETD-pNA, and LEHD-pNA, for caspase 3, 8, and 9, respectively (R&D System, Minneapolis, MN, USA). Concisely, the drug treated prostate cancer cells were harvested and washed by PBS for three times. Cell pellet was lysed on ice for 10 min, and then samples were centrifuged at 10,000 x g for 5 min. Protein content in the supernatants was measured using the Bradford reagent. Extracts were diluted to 4 mg/ml with dilution buffer, and loaded with 50 ml for each reaction well of 96-well microplate. Each reaction also required 50 ml of 2 x reaction buffer containing 10 mM of dithiothreitol and 5 ml of caspase colorimetric substrate. After 2-h incubation at 37℃, absorbance was measured at 405 nm on a microplate reader. Results are expressed as absorbance percent relative to control group (100%). Each experimental condition was repeated by three times.
Immunoblotting Assessment
After culture under the treatment of bufalin or cinobufagin at certain time points, cells were harvested and washed twice by ice-cold PBS. Cells were lysed in RIPA buffer (50 mM Tri-HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, l mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1mM Na-orthovanadate, 1 mM NaF) for 30-min on ice. The lysate was centrifuged at 14,000×g in 4℃ for 15 min, and the supernatant was collected. Equal amount of cell extract proteins (50-100 mg) were subjected to 10% (caspase 9) or 15% (caspase 3 and 8) SDS- polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH, USA). Membranes were incubated in blocking solution (5% dry milk in TBST containing 20 mM Tris-HCl, 135 mM NaCl, 0.1 % Tween 20, pH 7.6) followed by incubation with primary antibody overnight. The following primary antibodies were used as 1 mg/ml: caspase 3 from Imgenex (San Diego, CA, USA); caspase 8 from BioVion (Mountain View, CA, USA), caspase 9 from Medical and Biological Labortories (Nagoya, Japan); or β-actin from Sigma (St. Louis, MO, USA). After washing for three times by TBST, the blot was incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:20,000, Promega Corporation, Madison, WI, USA), and proteins were visualized using enhanced chemiluminescence detection (ECL, Western blotting reagents, Amersham International, Bucks, UK).
Isolation of Cytosol and Mitochondrial Fractions
Release of cytochrome c from mitochondria and translocation of bax to mitochondria was measured by immunoblotting essentially as described previously (16). Briefly, drug treated cancer cells were collected and washed twice by ice-cold PBS. The cell pellet was resuspended in 1 ml of extraction buffer (20 mM K+-Hepes, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2 · 6H2O, 0.1 mM EDTA, 1 mM EGTA, and protease inhibitors). Cells were lysed by 40 passages through a 26-gauge needle, and homogenates were centrifuged at 1000 x g for 5 min. The supernatant was again centrifuged at 10,000 x g for 15 min following the separation of supernatant (cytosolic fraction) from pellet (mitochondrial fraction). Cytosolic fraction was then concentrated to 50-100 ml using 10-kDa molecular mass centrifugal concentration device (microcon YM-10, Millipore Co., Bedford, MA, USA) according to the manufacturer’s instructions. The resulting mitochondrial pellets were resuspended in 50 ml of cell lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton, 1 mM phenylmethylsulfonyl fluride, and protease inhibitor mixture). These fractions were separated on 15% SDS-polyacrylamide electrophoresis gels with an equal amount of protein loaded onto each lane as determined by Bradford assay reagent. Cytochrome c and bax were detected by mouse monoclonal antibody at dilution of 1:400 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Each experimental condition was replicated by three times.