INHIBITORY EFFECTS OF DIGITALIS ON THE PROLIFERATION OF ANDROGEN DEPENDENT AND INDEPENDENT PROSTATE CANCER CELLS

0022-5347/01/1665-1937/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 166, 1937–1942, November 2001 Printed in U.S.A.

INHIBITORY EFFECTS OF DIGITALIS ON THE PROLIFERATION OF ANDROGEN DEPENDENT AND INDEPENDENT PROSTATE CANCER CELLS JIUN-YIH YEH, WILLIAM J. HUANG, SHU-FEN KAN

AND

PAULUS S. WANG*

From the Department and Graduate Institute of Physiology, School of Life Science, Department of Urology, School of Medicine, National Yang-Ming University and Division of Urology, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China

ABSTRACT

Purpose: Digitalis or cardiac glycosides have been noted to induce tumor static or oncolytic effects in various types of cancer. We evaluated the effects and underlying mechanisms of cardiac glycosides, including digoxin, digitoxin and ouabain, on the proliferation of hormone dependent and independent prostate cancer cell lines. Materials and Methods: Cell proliferation of the 3 human prostate cancer cell lines LNCaP, DU145 and PC3 was measured by 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetralozium bromide (Sigma Chemical Co., St. Louis, Missouri) colorimetric assay. The cytotoxic effects of digitalis on prostate cancer cells were determined by lactate dehydrogenase measurements of the culture medium. Intracellular Ca2⫹ was measured by a dual wavelength spectrometer system. The percent of apoptotic cells after digitalis treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and flow cytometry. Results: Digoxin, digitoxin and ouabain significantly inhibited the proliferation of LNCaP, DU145 and PC3 cells at a dose of 1 or 10 ␮M. after 1 to 4 days of culture. Cytotoxicity of digitalis on the DU145 and LNCaP cells was dose dependent but cytotoxicity was not obvious in PC3. Digitalis (1 ␮M.) significantly increased intracellular Ca2⫹ in LNCaP and DU145 after 12 hours of culture but PC3 cells needed a 24-hour treatment to show any effect. In the apoptosis measurement digitalis at a dose of 1 and 10 ␮M. also significantly increased the percent of apoptotic cells in the LNCaP, DU145 and PC3 cell lines. Normal control human glomerular epithelial cells showed no response to digitalis treatment at all tested doses. Conclusions: Digitalis may inhibit the proliferation of prostate cancer cell lines, although the 3 cell lines showed varied sensitivity to digitalis. These effects are possibly the result of a mechanism involving sustained elevation of the concentration of intracellular Ca2⫹ and of apoptosis. KEY WORDS: prostate, prostatic neoplasms, digitalis, apoptosis, calcium

Prostate carcinoma is the most common malignancy and age related cause of cancer death worldwide. Furthermore, its prevalence has increased progressively in recent decades.1, 2 Localized prostate carcinoma is potentially curable by a radical operation or definitive radiotherapy. However, metastatic prostate carcinoma can only be effectively controlled by hormone manipulation since the prostate is an androgen dependent organ. Since its beginning more than 60 years ago, hormone manipulation has been the mainstay of treatment for advanced prostate cancer. It offers many options, including surgical castration and other medical androgen depletion strategies. However, half of the patients have a disease flare 18 to 24 months after any type of hormone therapy. This condition is known as hormone refractory prostate cancer or stage D3 disease. Usually a downhill clinical course follows and patients die at an average of 6 months after the disease flare. Although many therapeutic protocols have been proposed, none currently available has proved dramatically effective.3–5 An extensive search for other potential types of management is now under way but the negative aspect of newly developed therapy is drug resistance or

toxic side effects.6 – 8 Some recent preliminary investigations have attempted to apply cardiac glycosides for prostate carcinoma therapy.9, 10 Such investigations imply that cardiac glycosides are potentially useful for treating hormone refractory prostate cancer. Digoxin, a purified digitalis preparation and a glycosides derived from the plant Digitalis lanata (foxglove), has been popular for heart failure therapy and atrial dysrhythmia for more than 100 years.11 Digoxin has been valued as a potent and highly selective Na⫹-K⫹-adenosine triphosphatase inhibitor. It is capable of elevating intracellular Ca2⫹ to sustained levels to boost myocardial contractility or inotropism.12 Toxin induced cell killing or cell apoptosis are processes provoked by sustained elevation of cytosolic Ca2⫹.13, 14 Therefore, manipulation aimed at increasing the concentration of intracellular Ca2⫹ may induce necrosis or apoptosis, or enhance the concentration of intracellular Ca2⫹. Thus, it is logical to treat malignant neoplasms, including hormone refractory prostate cancer, with digitalis.15 We evaluated the effects of digitalis, including digoxin, digitoxin and ouabain, on the proliferation, cytotoxicity, change in intracellular Ca2⫹ and appearance of apoptosis in the androgen dependent prostate cancer cell line LNCaP16 and androgen independent prostate cancer cell lines DU14517 and PC3.18

Accepted for publication May 4, 2001. Supported by Grant VGH 89-398-11. * Requests for reprints: Department and Graduate Institute of Physiology, School of Life Science, National Yang-Ming University, Taipei, Taiwan, Republic of China 11221. 1937

1938

INHIBITORY EFFECTS OF DIGITALIS ON PROSTATE CANCER MATERIALS AND METHODS

Prostate cancer cell line culture. We used the prostate cancer cell lines LNCaP (CCRC60088) (Culture Collection and Research Center, Food Industry Research and Development Institute, Taiwan, Republic of China as well as DU145 and PC3 cell lines (American Type Culture Collection, Rockville, Maryland). LNCaP was cultured with its standard RPMI 1640 medium (Gibco Laboratories, Grand Island, New York) containing 100 units per ml. potassium penicillin G (Sigma Chemical Co.), 100 ␮g. per ml. streptomycin sulfate (Sigma Chemical Co.) and 10% fetal calf serum (Kibbutz Beit, Haemek, Israel). DU145 and PC3 were cultured in 5% carbon dioxide/95% air at 37C with standard Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Laboratories) containing 100 units per ml. potassium penicillin G, 100 ␮g. per ml. streptomycin sulfate and 10% fetal calf serum. Normal human cell culture. Normal human glomerular epithelial cells were retrieved from a normal renal cortex resected from a tumor bearing kidney. These cells served as normal control human cells. They were plated in the same concentration and were treated with digitalis in conditions similar to those of the prostate cancer cell lines. The conditions of cell cytotoxicity, change in intracellular Ca2⫹ and appearance of apoptosis were also observed. Cell proliferation assessment. The modified colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done to quantify cell proliferation.19 Living cells reduced the yellow MTT to blue formazen, which was soluble in dimethyl sulfoxide (Wako, Osaka, Japan). The intensity of blue staining in the culture medium was proportional to the number of cells alive at analysis. The MTT assay used was similar to that described previously.20 Briefly, cells were incubated in 96-well microplates (Falcon, Franklin Lakes, New Jersey) for 24 hours as a pre-incubation process. Cells were plated at 2,000 cells per 200 ␮l. per well with Dulbecco’s modified Eagle’s medium or RPMI 1640 supplemented with 10% fetal calf serum. The culture medium was then removed and replaced by medium containing various concentrations of digoxin, digitoxin or ouabain (0, 0.01, 0.1, 1 or 10 ␮M.). After 24, 48, 72 or 96 hours the medium was removed and replaced by 50 ␮l. 1 mg./ml. MTT solution (Sigma Chemical Co.) in DMEM or RPMI 1640. After a further 4-hour incubation period the plates were centrifuged at 400⫻ gravity for 5 minutes. The MTT solution was again replaced by 50 ␮l. dimethyl sulfoxide and the plates were shaken for 3 minutes. The optical density of each well was determined using a microplate reader (Dynatech Laboratories, Chantilly, Virginia) at a wavelength of 570 nm. with a reference wavelength of 630 nm. Each experiment was performed in 6 preparations and repeated 4 times. Cytotoxicity assessment by lactate dehydrogenase assay. Cytotoxicity was measured using a cytotoxicity detection kit (Boehringer-Mannheim, Mannheim, Germany). The assay mechanism detects the lactate dehydrogenase activity in the culture medium. Oxidation of lactate to pyruvate results in increased nicotinamide adenine dinucleotide formation.21 Briefly, cells were incubated in 96-well microplates at 5,000 cells per well with DMEM or RPMI 1640 supplemented with 10% fetal calf serum. After 24 hours the culture medium was removed and replaced by medium containing various concentrations of digoxin, digitoxin or ouabain (0, 0.01, 0.1, 1 or 10 ␮M.). The wells for a high control condition were added to the medium containing 1% Triton X-100 (Sigma Chemical Co.) to determine total cellular lactate dehydrogenase. After a 24hour treatment 50 ␮l. supernatant per well were transferred into the corresponding wells of an optically clear, 96-well flat bottom microplate. To determine lactate dehydrogenase activity in the supernatants 50 ␮l. reaction mixture were added per well and incubated for 20 minutes in the dark at room temperature. Optical density in each well was determined by

a microplate reader (Dynatech Laboratories) at a wavelength of 490 nm. with a reference wavelength of 630 nm. Each experiment was performed in 6 preparations. Results were expressed as the percent of lactate dehydrogenase leakage. Measuring intracellular calcium. The 3 prostate cancer cell lines were treated with digoxin, digitoxin or ouabain (0.1 or 1 ␮M.) for 12 or 24 hours (PC3 only). The cells were harvested by the culture medium DMEM or by RPMI-1640 with 10% fetal calf serum and washed twice with the same culture medium. Cell suspension (1 ml. containing 5 ⫻ 106 cells) was loaded with 6 ␮g. fura2/acetoximethyl ester dissolved in 6 ␮l. dimethyl sulfoxide. They were incubated in the dark for 30 minutes at 37C. After extensive washing 1 ⫻ 106 cells were re-suspended in 2.5 ml. loading buffer, consisting of 152 mM. NaCl, 1.2 mM. MgCl2, 2.2 mM. CaCl2, 4.98 mM. KCl and 10 mM. Hepes. Fluorescence emission at 505 nm. was monitored at 37C by a dual wavelength spectrometer system with excitation at 340 and 380 nm. Free intracellular Ca2⫹ was calculated by the method of Grynkiewicz et al22 from the ratio of fluorescence intensity obtained every 1 second with a dissociation constant of 135 nM. Dye was considered saturated after lysis with digitonin at a final concentration of 0.16 mM. Minimum fluorescence was determined by adding 0.5 ml. egtazic acid (Sigma Chemical Co.) to the final concentration of 8 mM. Apoptosis detection. An apoptosis detection kit (Promega Corp., Madison, Wisconsin) was used to measure fragmented DNA by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling. Briefly, cells were pretreated with digoxin, digitoxin or ouabain (0, 0.1, 1 or 10 ␮M.) for 36 hours. The cells were harvested and washed with phosphate buffered saline (PBS) twice by centrifugation at 300 ⫻ gravity at 4C, re-suspended in 0.5 ml. PBS and fixed by adding 5 ml. 0.1% methanol-free formaldehyde for 20 minutes on ice. The cells were centrifuged at 300 ⫻ gravity for 10 minutes at 4C and re-suspended in 5 ml. PBS. The cells were washed again and re-suspended in 0.5 ml. PBS. Cell suspensions were added with 5 ml. 70% cold ethanol and maintained at ⫺20C. After at least 4 hours the cells were centrifuged, re-suspended in 1 ml. PBS and transferred into 1.5 ml. microcentrifuge tubes. After centrifugation at 300 ⫻ gravity for 10 minutes the supernatant was removed and the cells were resuspended in 80 ␮l. equilibration buffer containing 200 mM. potassium cacodylate, 25 mM. tris-HCl, 0.2 mM. dithiothreitol, 0.25 mg./ml. bovine serum albumin and 2.5 mM. cobalt chloride before incubation at room temperature for 5 minutes. The cells were centrifuged at 300 ⫻ gravity and the supernatant was replaced with 50 ␮l. terminal deoxynucleotidyl transferase incubation buffer mixture composed of 45 ␮l. equilibration buffer, 5 ␮l. nucleotide mix (50 ␮M. fluorescein-12-deoxyuridine triphosphate, 100 ␮M. deoxyadenosine triphosphate, 10 mM. tris-HCl and 1 mM. ethylenediaminetetraacetic acid) and 1 ␮l. (25 units) terminal deoxynucleotidyl transferase enzyme. After 60 minutes of incubation at 37C under light protection the reaction was terminated by adding 1 ml. 20 mM. ethylenediaminetetraacetic acid and the cells were centrifuged at 300 ⫻ gravity. The supernatant was removed and the cells were resuspended in 1 ml. 0.1% Triton X-100 solution in PBS containing 5 mg./ml. bovine serum albumin. The process was repeated once. After centrifugation at 300 ⫻ gravity for 10 minutes the supernatant was removed and the cell pellet was re-suspended in 0.5 ml. propidium iodide (Sigma Chemical Co.) solution (5 mg./ml. in PBS) containing 250 ␮g. deoxyribonuclease-free ribonuclease A. The cells were incubated at room temperature for 30 minutes in the dark and analyzed by EPICS flow cytometer (Beckman-Coulter, Corp., San Diego, California) with the excitatory argon laser at a wavelength of 488 nm. The green fluorescence of fluorescein2-deoxyuridine triphosphate and red fluorescence of pro-

INHIBITORY EFFECTS OF DIGITALIS ON PROSTATE CANCER

1939

pidium iodide were measured at 520 and 620 nm., respectively. Statistical analysis. For all groups data are presented as the mean plus or minus standard error of the mean (SEM). Statistical analysis was performed by 1-way analysis of variance followed by the Dunnett multiple range test of the difference in group means compared with control means.23 RESULTS

Effect of digitalis on the proliferation of prostate cancer cell lines. Figure 1 shows the effects of digitalis on the proliferation of prostate cancer cell lines. Proliferation status was assessed by MTT assay. The proliferation of LNCaP was significantly inhibited by digoxin, digitoxin or ouabain after treatment for 1 to 4 days (p ⬍0.01). Notably at 1 or 10 ␮M. all types of digitalis appeared toxic to LNCaP cells. The proliferation of DU145 was only inhibited by digoxin at 1 and 10 ␮M., by digitoxin at 10 ␮M., and by ouabain at 0.1, 1 and 10 ␮M. after treatment for 1 to 4 days (p ⬍0.01). The proliferation of PC3 was decelerated by digoxin and digitoxin at 1 and 10 ␮M. but significantly inhibited by ouabain at 0.1, 1 and 10 ␮M. after treatment for 1 to 4 days (p ⬍0.01). Cytotoxicity of digitalis in prostate cancer cell lines. The effects of cytotoxicity were measured by lactate dehydrogenase assay after a 24-hour digitalis treatment (fig. 2). All 3 digitalis types achieved significant cytotoxic effects corresponding to the amount of digitalis administered. However, effects seemed to be more prominent in the hormone dependent LNCaP cells since the lower tested dose of 0.1 ␮M. showed significant effects. Of the hormone independent cell lines DU145 showed cytotoxicity only at higher doses of digitalis (1 or 10 ␮M.), while PC3 showed negligible effects at higher doses. These outcomes correlated well with the findings of proliferation studies, in which LNCaP cell populations

FIG. 1. Mean effects plus or minus SEM of digoxin, digitoxin and ouabain on proliferation of LNCaP, DU145 and PC3 cells after incubation period of 1 to 4 days.

FIG. 2. Mean effects of cytotoxicity plus or minus SEM of digoxin, digitoxin and ouabain on LNCaP, DU145 and PC3 cells after 24 hours of treatment. Asterisk indicates p ⬍0.05 and double asterisks indicate p ⬍0.01 versus concentration at 0.01 ␮M.

progressively decreased with a corresponding increase in drug concentration or incubation time (fig. 1). Effects of digitalis on intracellular Ca2⫹ in prostate cancer cells. The change in intracellular Ca2⫹ in cancer cells was assessed after 12-hour digitalis treatment. The intracellular

FIG. 3. Mean effects plus or minus SEM of digoxin, digitoxin and ouabain on intracellular calcium change in LNCaP, DU145 and PC3 cells after 12 hours of treatment. Asterisk indicates p ⬍0.05 and double asterisks indicate p ⬍0.01 versus value at 0 M.

1940

INHIBITORY EFFECTS OF DIGITALIS ON PROSTATE CANCER

Ca increase was evident at higher doses (0.1 to 1 ␮M.) of digitalis in LNCaP and DU145 cells. PC3 responded only to a high dose (1 ␮M.) of ouabain (p ⬍0.05, fig. 3). LNCaP cells seems to be more sensitive than DU145 or PC3 cells to digitalis in terms of increasing intracellular Ca2⫹. Doses of digoxin to the LNCaP cells at 0.1 ␮M. also induced a significant intracellular Ca2⫹ increase (p ⬍0.01). Since PC3 showed the most resistance to the digitalis challenge in terms of intracellular Ca2⫹ change, we treated PC3 with an extended period of incubation to determine whether any change in intracellular Ca2⫹ would occur. Therefore, incubation time was extended to 24 hours. The intracellular Ca2⫹ increase in PC3 was consequently significant in terms of the response to all 3 types of digitalis tested (p ⬍0.05 for 0.1 and p ⬍0.01 for 1 ␮M., fig. 4). Apoptosis of prostate cancer cells induced by digitalis. We determined apoptosis after a 36-hour digitalis treatment. Figure 5 shows a representative contour diagram of fluorescein-deoxyuridine triphosphate/propidium iodide flow cytometry of LNCaP, DU145 and PC3 cells after digoxin, digitoxin or ouabain treatment at various doses. The apoptotic population increased with an increase in treatment drugs. The percent of apoptotic cells were further transformed into a bar chart for statistical analysis (fig. 6). After digitalis treatment significant apoptosis was induced in LNCaP and DU145 by adding 1 or 10 ␮M. digitalis. LNCaP cells were more sensitive than the DU145 or PC3 cells to digitalis treatment. They responded to all 3 digitalis types tested at 1 or 10 ␮M. While DU145 cells showed varied sensitivity to all 3 digitalis types at 1 ␮M., ouabain produced the most evident effect. PC3 cells were the most inert of all cell lines. They showed no reaction to 1 ␮M. digitalis. As the drug concentration increased to 10 ␮M., PC3 showed a moderate response of an average of 20% of apoptotic cells to digitoxin or ouabain but no response to digoxin. 2⫹

DISCUSSION

Prostate carcinoma is a common malignancy and age related cause of cancer death in male Americans.24, 25 The inFIG. 5. Contour diagram of fluorescein-deoxyuridine triphosphate/propidium iodide (PI) flow cytometry of LNCaP, DU145 and PC3 cells after digoxin, digitoxin and ouabain treatment at various doses. Cell populations at upper left and right quadrants of each panel represent apoptotic cells.

FIG. 4. Mean effects plus or minus SEM of digoxin, digitoxin and ouabain on intracellular calcium change in PC3 cells after 24 hours of treatment. Asterisk indicates p ⬍0.05 and double asterisks indicate p ⬍0.01 versus value at 0 M.

cidence of prostate cancer in Asian men has also increased rapidly in recent years.26 Although an early diagnosis contributes to the potential cure of localized prostate cancer, many patients still have from metastatic disease at presentation. Since the prostate is an androgen dependent organ, androgen ablation continues to be the mainstay of treatment. There are many methods of androgen ablation for metastatic prostate cancer, including surgical castration, oral estrogen therapy, luteinizing hormone-releasing hormone analogue depot injection and antiandrogen therapy. Other than hormone manipulation some systemic treatments, such as chemotherapy or combined therapy, have also been proposed despite a limited disease response.27 However, prostate carcinoma may gradually become refractory to hormonal therapy after a period of treatment. This so-called hormone refractory status or stage D3 disease is also believed to be resistant to chemical and any other currently available therapies, and patients usually survived no more than 6 months. Therefore, searching for newer treatment strategies for advanced prostate cancer is a priority task. Epidemiological studies had shown that digitalis has anticancer effects.28 Patients with breast cancer treated with digitalis have been found to have a lower death rate compared with those not treated with digitalis.28 Five years after

INHIBITORY EFFECTS OF DIGITALIS ON PROSTATE CANCER

FIG. 6. Mean percent plus or minus SEM of apoptotic cells in the LNCaP, DU145 and PC3 cells after 36-hour digoxin, digitoxin and ouabain treatments, as calculated from cell populations in upper left and right quadrants of each panel (fig. 5).

mastectomy the cancer recurrence rate in controls not given digitalis was 9.6-fold that of patients treated with digitalis.29 According to recent reports the cardiac glycoside ouabain can sensitize malignant cells to irradiation.30 The results of this study show that digitalis, including digoxin, digitoxin and ouabain, exert significant inhibitory effects on the proliferation of all tested prostate cancer cell lines, namely LNCaP, DU145 and PC3. The inhibitory effect is dose dependent and time related (fig. 1). In LNCaP cells digoxin, digitoxin and ouabain effectively inhibited proliferation at doses of 0.1 to 10 ␮M. Ouabain had the strongest inhibitory effect. The anti-proliferation effects of digitalis on DU145 and PC3 cells were similar except 1 ␮M. digitoxin was effective in PC3 but not in DU145 cells. Of the 3 types of digitalis ouabain was more effective for inhibiting the proliferation of prostate cancer cells than digoxin or digitoxin. In regard to digitalis sensitivity overall LNCaP cells were the most sensitive. At 1 and 10 ␮M. digoxin, digitoxin and ouabain not only inhibited the proliferation of LNCaP, but also decreased the cell number compared with controls at day 0. These results indicate that digitalis has a cytotoxic rather than a cytostatic effect on the LNCaP cell line. After a 24hour treatment digitalis produced a cytotoxic effect on prostate cancer cells (fig. 2). The most sensitive cell line was LNCaP, followed by DU145 and PC3. Lactate dehydrogenase release from the 3 cell lines induced by digitalis showed a dose dependent relationship. These results correlated well with MTT assay results. After a 12-hour incubation with 0.1 and 1 ␮M. digitalis the LNCaP and DU145 cells showed a tremendous increase in intracellular Ca2⫹ (fig. 3). PC3 responded with increased intra-

1941

cellular Ca only to 1 ␮M. ouabain. After an extended treatment period of 24 hours digoxin, digitoxin and ouabain increased intracellular Ca2⫹ in PC3 cells and the increase was also dose dependent (fig. 4). These data correlated with the cytotoxicity effect of digitalis on prostate cancer cells. The results suggested that digitalis inhibits the proliferation of prostate cancer cell lines through a mechanism of cytotoxicity effects induced by a sustained elevation in intracellular Ca2⫹. It is well known that digitalis has the ability to inhibit the activity of Na⫹/K⫹-adenosine triphosphatase and lead to increased intracellular Ca2⫹.12 Deregulation of intracellular Ca2⫹ results in activation of a number of intracellular pathways, such as a change in cellular structure or gene expression. A sustained elevation in intracellular Ca2⫹ may also activate various hydrolytic enzymes, including proteases, nucleases and lipase,31 which have been implicated as effectors of Ca2⫹ elicited toxicity. Thus, these changes may have a critical role in cellular toxicity.14 Activated hydrolytic enzymes may lead to exaggerated energy expenditure, impair energy production, initiate cytoskeletal degradation, decrease phospholipids, disrupt membrane and cytoskeletal proteins, and nuclear chromatin, and ultimately result in cell death. The results of apoptosis studies correlated well with cytotoxicity data in the lactate dehydrogenase assay, although they needing a long incubation period of 36 hours (figs. 5 and 6). We observed apoptosis after a shorter exposure time of 12 or 24 hours but still no apoptotic cells appeared. Together with the results of intracellular Ca2⫹ studies it may be proposed that digitalis induced apoptosis occurs after an elevation in intracellular Ca2⫹. In this study intracellular Ca2⫹ in LNCaP, DU145 and PC3 cells was raised markedly about 2 to 4-fold by adding digitalis. This sustained elevation in intracellular Ca2⫹ possibly may have led to cell toxicity and cell death. The cell death promoting activity of cardiac glycosides appears cell-type selective. In this study androgen dependent LNCaP cells were more susceptible to digitalis than androgen independent DU145 or PC3 cells. McConkey et al studied cardiac glycoside susceptibility in PC3 subclones and found that various subclones showed varied apoptosis induction responses.9 They attributed the results to a possible delay in mitochondria cytochrome C release in a more resistant subclone. To our knowledge whether the different sensitivity of LNCaP, DU145 and PC3 cells to digitalis were due to mechanisms involving intracellular calcium dynamics remains to be determined. Digitalis or cardiac glycosides are drugs with a narrow therapeutic safety range. Clinically cardiac glycoside intoxication is not uncommon with mild to severe symptoms, such as vomiting, cold sweat, bradycardia and convulsion or even syncope and death. Therefore, careful blood level monitoring and early detection of intoxication signs in patients on digitalis treatment cannot be overemphasized. In this study we used normal human cells (glomerular epithelial cells) as a normal control to verify the tumor specific anti-proliferation oncolytic effects, induction of increased intracellular Ca2⫹ and apoptosis effects of the tested digitalis. We found no particular change in evaluated criteria in normal human control cells after the digitalis challenge. Although glomerular epithelial cells cannot represent each type of human cells, it is evident that digitalis has certain tumor or organ (prostate) specific effects. Safer in vitro studies in other normal human cells are needed. In future in vivo studies careful titration of doses of digitalis should be ensured. In addition to the cardiac effects studies of patients receiving long-term digoxin therapy have shown changes in plasma testosterone and luteinizing hormone.32 In previous experiments we have also found that digoxin has an inhibitory effect on testosterone secretion through mechanisms involving decreased cyclic adenosine monophosphate production and cytochrome P450scc activity in rat testicular interstitial cells.33 Since digoxin has inhibitory effects on the production 2⫹

1942

INHIBITORY EFFECTS OF DIGITALIS ON PROSTATE CANCER

of testosterone in testis, digitalis may also interfere with the growth of prostate cancer cells in vivo by decreasing circulating plasma testosterone. These characteristics of cardiac glycosides may imply some synergistic effects of various mechanisms of prostate cancer treatment in vivo. CONCLUSIONS

Digitalis can directly inhibit the proliferation of the androgen dependent prostate cancer cell line LNCaP and the androgen independent prostate cancer cell lines DU145 and PC3. This effect may be achieved by an elevation in intracellular Ca2⫹ and by apoptosis. The 3 prostate cancer cell lines showed various degrees of sensitivity to digitalis treatment. Since cardiac glycosides are drugs with a narrow range of therapeutic safety, more detailed in vitro intoxication trials in various normal human cells are needed before wide range in vivo studies are started. Drs. C. R. Jan and Tony T. Wu, Veterans General Hospital, Kaohsiung provided technical assistance with the prostate cancer cell lines, and Dr. Ching-yuang Lin, Department of Pediatrics, Taipei Veterans General Hospital provided technical assistance with human glomerular epithelial cell culture. REFERENCES

1. Rhim, J. S. and Kung, H. F.: Human prostate carcinogenesis. Crit Rev Oncogen, 8: 305, 1997 2. Chang, C. K., Yu, H. J., Chan, K. W. et al: Secular trend and age-period cohort analysis of prostate cancer mortality in Taiwan. J Urol, 158: 1845, 1997 3. Abrahamsson, P. A.: Neuroendocrine differentiation in prostatic carcinoma. Prostate, 39: 135, 1999 4. Tilley, W. D., Buchanan, G., Hickey, T. E. et al: Mutations in the androgen receptor gene are associated with progression of human prostate cancer to androgen independence. Clin Cancer Res, 2: 277, 1996 5. Crawford, E. D., Eisenberger, M. A., McLeod, D. G. et al: A controlled trial of leuprolide with and without flutamide in prostatic cancinoma. N Engl J Med, 321: 419, 1989 6. Teicher, B. A., Kakeji, Y., Ara, G. et al: Prostate carcinoma response to cytotoxic therapy: in vivo resistance. In Vivo, 11: 453, 1997 7. Theyer, G., Schirmbock, M., Thalhammer, T. et al: Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines. J Urol, 150: 1544, 1993 8. Berlin, J. D., Propert, K. J., Trump, D. et al: 5-Fluorouracil and leucovorin, therapy in patients with hormone refractory prostate cancer: an Eastern Cooperative Oncology Group phase II study (E1889). Am J Clin Oncol, 21: 171, 1998 9. McConkey, D. J., Lin, Y., Nutt, L. K. et al: Cardiac glycosides stimulate Ca2⫹ increases and apoptosis in androgenindependent, metastatic human prostate adenocarcinoma cells. Cancer Res, 60: 3807, 2000 10. Haux, J.: Digitoxin is a potential anticancer agent for several types of cancer. Med Hypotheses, 53: 543, 1999 11. Withering, W.: An Account of the Foxglove, and Some of its Medical Use: With Practical Remarks on Dropsy, and Other Diseases. Birmingham, United Kingdom: Swinney, p. 232, 1785 12. Schmidt, T. A., Allen, P. D., Colucci, W. S. et al: No adaptation to

13. 14. 15.

16. 17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

digitalisation as evaluated by digitalis receptor (Na, K-ATPase) quantification in explanted hearts from donor without heart disease and from digitalised recipients with end-stage heart failure. Am J Cardiol, 71: 110, 1993 Jones, D. P., McConkey, D. J., Nicotera, P. et al: Calciumactivated DNA fragmentation in rat liver nuclei. J Biol Chem, 264: 6398, 1989 Trump, B. F. and Berezesky, I. K.: Calcium mediated cell injury and cell death. FASEB J, 9: 219, 1995 Furaya, Y., Lundmo, P., Short, A. D. et al: The role of calcium, pH and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res, 44: 3522, 1984. Horoszewicz, J. S., Leong, S. S., Kawinski, E. et al: LNCaP model of human prostatic carcinoma. Cancer Res, 43: 1809, 1983 Stone, K. R., Mickey, D. D., Wunderli, H. et al: Isolation of a human prostate carcinoma cell line (DU145). Int J Cancer, 21: 274, 1978 Kaighn, M. E., Narayan, K. S., Ohnuki, Y. et al: Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol, 17: 16, 1979 Chung, T. D., Yu, J. J., Spiotto, M. T. et al: Characterization of the role of IL-6 in the progression of prostate cancer cells. Prostate, 38: 199, 1999 Janssen, T., Darro, F., Petein, M. et al: In vitro characterization of prolactin-induced effects on proliferation in the neoplastic LNCaP, DU145, and PC3 models of the human prostate. Cancer, 77: 144, 1996 Chen, L., Zhang, B. H. and Harris, D. C.: Evidence suggesting that nitric oxide mediates iron-induced toxicity in cultured proximal tubule cells. Am J Physiol, 274: F18, 1998 Grynkiewicz, G., Poenie, M. and Tsien, R. Y.: A new generation of Ca2⫹ indicators with greatly improved fluorescence properties. J Biol Chem, 260: 3440, 1985 Steel, R. D. and Torrie, J. H.: Principles and Procedures of Statistics, 2nd ed. New York: McGraw-Hill, 1980 Lange, P. H.: Early detection for prostate cancer. J Natl Cancer Inst, 83: 1199, 1991 Stearns, M. E. and Wang, M.: Type IV collagenase (M(r)72,000) expression in human prostate: benign and malignant tissue. Cancer Res, 53: 878, 1993 Hsing, A. W., Devesa, S. S., Jin, F. et al: Rising incidence of prostate cancer in Shanghai, China. Cancer Epidemiol Biomarkers Prev, 7: 83, 1998 Hsieh, T. C. and Wu, J. M.: Effects of fenretinide (4-HPR) on prostate LNCaP cell growth, apoptosis, and prostate-specific gene expression. Prostate, 33: 97, 1997 Stenkvist, B.: Is digitalis a therapy for breast carcinoma? Oncol Rep, 6: 493, 1999 Stenkvist, B., Pengtsson, E., Dahlqvist, B. et al: Cardiac glycisides and breast cancer, revisited. N Engl J Med, 306: 484, 1982 Verheye-Dua, F. and Bohm, L.: Na⫹-K⫹-ATPase inhibitor, ouabain accentuates irradiation damage in human tumour cell lines. Radiat Oncol Investig, 6: 109, 1998 Nicotera, P. and Orrenius, S.: The role of calcium in apoptosis. Cell Calcium, 23: 173, 1998 Stoffer, S. S., Hynes, K. M., Jiang, N. S. et al: Digoxin and abnormal serum hormone levels. JAMA, 225: 1643, 1973 Lin, H., Wang, S. W., Tsai, S. C. et al: Inhibitory effect of digoxin on testosterone secretion through mechanisms involving decreases of cyclic AMP production and cytochrome P450scc activity in rat testicular interstitial cells. Br J Pharmacol, 125: 1635, 1998