Disruption of Mitochondrial Function by Suramin Measured by Rhodamine 123 Retention and Oxygen Consumption in Intact DUI45 Prostate Carcinoma Cells I

[CANCER RESEARCH 52, 6953-6955, December 15, 1992] Advances in Brief

Randall P. Rago, Peter C. Brazy, and George Wiiding 2 William S. Middleton Veteran's Hospital [R. P. R., P. C. B., G. W.], University of Wisconsin Comprehensive Cancer Center [R. P. R., G. W.], and Department of Medicine [P. C. B.], University of Wisconsin Hospitals and Clinics, Madison, Wisconsin 53792

Abstract Oxygen consumption and ATP were measured before and after suramin addition in cell suspensions. Suramin, an antiparasitic drug, has shown antitumor activity in humans. This may occur in part through disruption of energy balance, Materials and Methods which is believed to be part of its antiparasitic action. Suramin disrupts mitochondrial function in intact DUI45 prostate carcinoma cell mono- DU145 human prostate carcinoma cells were grown at 37~ and 5% layers as seen by its causing the release of rhodamine 123 from CO2 to approximately 75% confluency in DMEF53 and were prestained cells beginning at about 10 ~tM in 96-well microtiter plates trypsinized from plates with two rinses with of 0.5% trypsin. measured with a fluorescent plate scanner. This effect was similar to the For rhodamine 123 studies 20,000 DU145 cells/well were seeded in ionophore carbonyl cyanide m-chlorophenylhydrazone, dissolved in eth- 100 #! of DMEF5 into 96-well microculture plates on day -1. Twenty- anol at 0.01 N and indicates that suramin acts as a respiratory poison or four h later cells were incubated with 10 #g/ml of rhodamine 123 for an ionophore. This effect was confirmed by studies of oxygen consump- 10 min, and then the wells were emptied by blotting the microtiter tion with a Clark oxygen electrode and cellular ATP content which plates on toweling. The wells were rinsed twice with phosphate-buffered demonstrated uncoupling of oxidative phosphorylation by 100 #M saline and emptied each time. The cells were incubated with suramin in suramin, a clinically achievable plasma drug level. DMEF5 at final concentrations of 10, 100, and 1000 #M suramin or 1, 10, and 100 #M CCCP. At time points of interest the wells were again Introduction emptied and rinsed with phosphate-buffered saline. The retained fluo- Suramin, a polysulfonated naphthylurea, is an antitrypano- rescence was measured after lysing cells by the addition of 100 #1 of distilled water to the wells. The fluorescence was measured with a somal agent that has been used to treat African sleeping sick- 96-well fluorescent plate scanner (Fluoroskan II; Flow Laboratories, ness and onchocerciasis since the 1920s (1). Suramin is known McLean, VA) with filters reading fluorescein. The fluorescence in the to inhibit respiration and glycolysis in trypanosomes, and this is presence of suramin or CCCP is expressed as a percentage of corre- believed to be part of the antiproliferative mechanism of sponding controls which contained no suramin or CCCP. suramin in trypanosomes (2). Suramin has been shown to have Oxygen consumption Qo2 were determined by standard methods antiproliferative effects in human tumors in vivo and in vitro (9) with a Clark-type oxygen electrode (Yellow Springs Instrument Co., (3-5). Suramin is active in many in vitro assays and this has Yellow Springs, OH). DU 145 cells prepared as above were washed in a resulted in the proposal of numerous antiproliferative mecha- Krebs-Ringer bicarbonate medium that contained lactate (5 mM), ala- nisms. Part of the antiproliferative effect of suramin in tumors nine (1 mM), and malate (1 mM) as the metabolic substrates (no glu- may involve inhibition of energy balance, as in trypanosomes. cose). The DUI45 cells were suspended in this medium at a concentration of 2-3 mg protein/1.7 ml. The suspension was oxygenated and Inhibition of respiration in isolated mammalian mitochondria preincubated for 20 min at 37~ and then placed in the closed, ther- (6) and inhibition of glycolysis in colon carcinoma cells (7) have mostated (37~ chamber (1.7 ml) for measurement of Qo2- A control been demonstrated with suramin. We have previously shown Qo2 was determined over the next 2 to 5 min. Then 100 #M suramin or that suramin inhibits mitochondrial activity in intact DU145 1 uM oligomycin followed by 100 #M suramin (final concentrations) was prostate carcinoma cells at concentrations which are clinically added to the chambers and Qo2 was determined. At the end of the achievable and active in humans (8). This was seen by inhibition observation period aliquots of the cell suspension were taken for the of tetrazolium conversion by mitochondrial dehydrogenases in measurement of protein (10) and ATP (11). The data are factored by the the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- amount of protein in the suspension. Time controls for DU145 cells mide assay, inhibition of rhodamine 123 mitochondrial uptake gave a linear rate of oxygen consumption over a 5- to 10-min observa- and retention by fluorescent microscopy, and toxic mitochon- tion period. Suramin was dissolved in saline and added to the suspension at a final concentration of 100 #M. Oligomycin was dissolved in drial changes seen by electron microscopy. In this article we 95% ethanol and was added to the suspension at a final concentration provide additional quantitative and confirmatory studies docu- of 1 #M. The experiments were performed on five to seven preparations menting the effect of suramin on mitochondria in intact DU 145 of DU 145 cells, with duplicate samples being run for each preparation. human prostate carcinoma cells. Cell monolayers with mitochondria prestained with rhodamine 123 were measured for Results and Discussion loss of rhodamine 123 fluorescence after suramin treatment. Rhodamine 123 is a fluorescent cation that localizes to mitochondria and its retention is dependent on the maintenance of Received 9/22/92; accepted 10/26/92. the negative electrochemical gradient across the mitochondrial The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accord- membrane. Rhodamine 123 release from prestained cells has ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grant CA-50590 and the Veterans Administration. 2 To whom requests for reprints should be addressed, at University of Wisconsin 3 The abbreviations used are: DMEF5, Dulbecco's modified Eagle's medium Comprehensive Cancer Center, K4/666 CSC, 600 Highland Ave., Madison, WI with 5% fetal calf serum; CCCP, carbonyl cyanide m-chlorophenylhydrazone dis- 53792. solved in ethanol at 0.01 M. 6953 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. SURAMIN UNCOUPLES RESPIRATION AND DISRUPTS MITOCHONDRIAL MEMBRANE POTENTIAL been shown to be caused by compounds that are respiratory Table 1 Acute effect of suramin on oxygen consumption by DU145 cells poisons or ionophores (12). The inhibitory effect of suramin on Aliquots of DU145 cell suspensions (2-3 mg protein/1.7 ml) were aerated with 95% 02/5% CO2 and preincubated for 30 min at 37~ without glucose to the retention of rhodamine 123 is shown in Fig. 1A. Suramin achieve steady state. Oxygen tensions of the suspensions were measured polaro- caused a dose-dependent and time course-related decrease in graphically with a Clark-type electrode and rates of oxygen consumption are mitochondrial retention of rhodamine 123 in a manner similar expressed as nmol/mg cellular protein/min. to the ionophore CCCP (Fig. 1B). The plots show that suramin Qo2 ATP is likely a weaker respiratory poison or ionophore than the Conditions (nmol/mg protein min)a (nmol/mgprotein)a ionophore CCCP. This effect was seen at less than 1 h and Control a 16.8 _+ 1.2 14.0 + 1.3 + Suramin, 100 #M 28.8 + 3.2 12.4 _+ 1.1 beginning at approximately 10 ~tM suramin. This concentration Oligomycin, 1 ~M/' 5.3 + 1.0 5.5 --+ 0.7 is less than one-tenth the concentration of suramin routinely + Suramin, 100 ~M 27.3 + 6.4 measured in plasma of patients in current Phase I trials. Equiv- a Valuesare the average _+ SD of n determinations.Each determinationwas the average of two samplesrun simultaneously. alent results were obtained in four other experiments. bn=7. The data for measurements of cell oxygen consumption rates Cn=5. and ATP content are given on Table 1. The control rate of Qo2 for DU145 cells was 16.8 _+ 1.2 nmol/mg protein/min. The The immediate effect of suramin contact with DU145 cells is addition of suramin acutely increased this rate to 170% without to "uncouple" oxidative phosphorylation by increasing mito- an increase in ATP content. In the presence of oligomycin, an chondrial respiration without an increase in ATP production. inhibitor of mitochondrial ATPase and oxygen-coupled respi- This modest "uncoupling" of oxidative phosphorylation may ration (13), Qo2 was reduced to 5.3 _+ 1.0 nmol/mg protein/min. deprive the cell of ATP. Dissipation of the mitochondrial mem- Addition of suramin acutely increased Qo2 to 515%. Increasing brane potential, seen with rhodamine 123 studies, also indicates oxygen consumption in the presence of oligomycin clearly in- that the cell could not generate ATP effectively. While it is not dicates that suramin behaves as an uncoupler of mitochondrial clear that these events would lead to cell death, ATP depriva- respiration in these cells. tion would likely be associated with antiproliferative effects. In our previous studies (8) we showed that even a high concentration of 1000 ~M suramin for 3 days had largely a static A effect on DU 145 cells. Greater than 50% of colonies in a colony formation assay could be counted 8 days after drug removal. 150 T 14r, J_ 0~0 1000 uM SURAMIN There was some irreversible toxicity seen, however, with long 130-"1 e---e 100 uM SURAMIN T A... ,,, 10 uM SURAMIN drug exposure. Also seen by electron microscopy were time- 1201- '=--& 0 SURAMIN dependent mitochondrial changes. After a 1-h exposure of ~p_. 1101- T T z~ 1001- l DU 145 cells to 100 #M suramin, the mitochondria swelled, with tO~o 90+ ~ ...... progressive toxic mitochondrial changes. Later disruption of cristae with intramitochondrial precipitates, followed by pro- gression to end-stage multivesicular bodies, was documented. 50 ~- 40 These findings were similar to those seen with two sulfonylurea n LI_ 30 compounds that caused uncoupling of oxidative phosphoryla- 20 10 tion in isolated murine liver mitochondria and swelling of mi- 0 I t I ! I I i t tochondria at 24 h in a colon carcinoma cell line (14). In all of 0 1 2 3 4 5 6 7 8 these studies it is unclear if mitochondrial changes or ATP HOURS deprivation accounts for cell death or is the major antiproliferative mechanism. These issues are the object of ongoing studies. All of our mitochondrial studies have been performed using B intact DU145 cells rather than cell preparations or isolated 150 o~o I00 uM CCCP 140 mitochondria. How suramin affects this intraceUular organeUe o---I 10 uM CCCP 130 A.-. A I uMCCCP and its function is uncertain. We hypothesize that suramin _1 120 A--A 0 CCCP affects mitochondria by interacting with the plasma mem- 110 100 brane and perhaps by affecting ion fluxes or second messenger z o 90 .L '- "/~.. systems. it. 80 ~\ "''''" .. 0 70 j. .. We have recently presented evidence that suramin can cause I-- Z 60 ~''.-- ...... a ...... a mitochondrial myopathy and Fanconi's syndrome in humans, tO 50 indicating a mitochondrial toxic effect in humans (15). These i,i 40 n 30 clinical entities of mitochondrial myopathy and Fanconi's syn- 20 drome are seen together in the De Toni-Fanconi-Debre (16, 17) 10 0 I I I I I ! ' ! I syndrome which is the congenital deficiency of mitochondrial 0 1 2 3 4 5 6 7 8 cytochrome c oxidase. This supports the concept that suramin

HOURS can inhibit mitochondrial activity in humans and this mecha- Fig. 1. Mitochondrialretention of rhodamine 123 in the presence of suramin nism may be involved in other known toxicities. Suramin has or CCCP. Twenty thousand DU145 cells/wellwere seeded in 100 ~1 of DMEF5 unique mechanisms which require further study to develop new into 96-wellmicroculture plates on day-1. Cellswere incubatedwith 10 ~g/ml of rhodamine 123 for 10 min, and then the cellswere rinsed twice with phosphate- therapeutic strategies and therapies and decrease toxicities. buffered saline. The cells were incubated with suramin in DMEF5 at final concentrations of 10, I00, and 1000/~Msuramin (,4) or 1, 10, and 100 #M CCCP (B) References for various lengths of time. At time points of interest the cellswere again rinsed, and retained fluorescencewas measured with a 96-wellfluorescent plate scanner 1. Steck, E. A. Suramin. In: The Chemotherapy of Protozoan Diseases, Vol. 2, after lysisof cells in distilledwater. pp.31-40. Washington, D.C.: Walter Reed Army Institute of Research, 1971. 6954 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. SURAMIN UNCOUPLES RESPIRATION AND DISRUPTS MITOCHONDRIAL MEMBRANE POTENTIAL

2. Fairlamb, A. H., Bowman, I. B. Uptake of the trypanocidal drug suramin by I0. Spector, T. Refinement of the Commassie blue method of protein quantita- bloodstream forms of Trypanosoma brucei and its effect on respiration and tion. Anal. Biochem., 86: 142-146, 1978. growth rate in vivo. Mol. Biochem. Parisitol., 1: 315-333, 1980. 11. Stanley, P. E., and Williams, S. G. Use of the liquid scintillation spectro- 3. Stein, C. A., LaRocca, R. V., Thomas, R., McAtee, N., and Myers, C. E. meter for determining adenosine triphosphate by luciferase enzyme. Anal. Suramin, an anticancer drug with a unique mechanism of action. J. Clin. Biochem., 29: 381-392, 1969. Oncol., 7: 499-508, 1989. 12. Johnson L. V., Walsh, M. L., Bockus, B. J., and Chen., L. B. Monitoring of 4. Myers, C., Cooper, M., Stein, C., LaRocca, R., Walther, M. M., Weiss, G., relative mitochondrial membrane potential in living cells by fluorescence Choyke, P., Dawson, N., Steinberg, S., Uhrich, M. M., Cassidy, J., Kohler, microscopy, J. Cell Biol., 88" 526-535, 1981. D. R., Trepel, J., and Linehan, W. M. Suramin: a novel growth factor an- 13. Lardy, H. A., Johnson, D., and McMurray, W. C. Antibiotics as a tool for tagonist with activity in hormone-refractory metastatic prostate cancer. J. metabolic studies. I. A survey of toxic antibiotics in respiratory, phosphory- Clin. Oncol., I0: 881-889, 1992. lative, and glycolytic systems. Arch. Biochem. Biophys., 78: 587-597, 1958. 5. Tkacuk, K., Eisenberger, V., Sinibaldi, V., Reyno, L., Jodrell, D., Jacobs, S., 14. Thakar, J. H., Chapin, C., Berg, R. H., Ashmun, R. A., and Houghton, P. J. Melink, T., Pearl, P., Aisner, J., Van Echo, D., and Egorin, M. Activity of Effect of antitumor diarylsulfonylureas on in vivo and in vitro mitochondrial suramin in prostate cancer observed in a Phase I trial. Proc. Am. Soc. Clin. structure and functions. Cancer Res., 51: 6286-6291, 1991. Oncol., I1: 618, 1992. 15. Rago, R. P., Sufit, R., Miles, J., Arzoomanian, R., Alberti, D., Tutsch, K., 6. Calcaterra, N. B., Vicario, L. R., and Roveri, O. A. Inhibition by suramin of Hutson, P., Spriggs, D., and Wilding, G. Suramin weakness due to Fanconi's mitochondrial ATP synthesis. Biochem. Pharmacol., 37: 2521-2527, 1988. syndrome and mitochondrial myopathy supports disruption of mitochondria 7. Fantini, J., Rognoni, J., Roccabianca, M., Pommier, G., and Marvaldi, J. or energy balance as mechanism of activity. Proc. Am. Soc. Clin. Oncol., 11: Suramin inhibits cell growth and glycolytic activity and triggers differentia- 279, 1992. don of human colic adenocarcinoma cell clone HT29-D4. J. Biol. Chem., 16. Van Biervliet, J. P., Bruinvis, L., Ketting, D., De Bree, P. K., Van Der 264: 10282-10286, 1989. Heiden, C., and Wadman, S. K. Hereditary mitochondrial myopathy with 8. Rago, R. P., Mitchen, J. M., Cheng, A. L., Oberly, T., and Wilding, G. lactic acidemia, a De Toni-Fanconi-Debre syndrome, and a defective respi- Disruption of cellular energy balance by suramin in intact human prostatic ratory chain in voluntary striated muscles. Pediatr. Res., 11: 1088-1093, carcinoma cells, a likely antiproliferative mechanism. Cancer Res., 51: 6629- 1977. 6635, 1991. 17. DiMauro, S., Mendell, J. R., Sahenk, Z., Bachman, D., Scarpa, A., Scofield, 9. Brazy, P. C., and Klotman, P. E. Increased oxidative metabolism in renal R. M., and Reiner, C. Fatal infantile mitochondrial myopathy and renal tubules from spontaneously hypertensive rats. Am. J. Physiol., 257:818-825, dysfunction due to cytochrome-c-oxidase deficiency. Neurology, 30: 795- 1989. 804, 19~0.

6955 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. Disruption of Mitochondrial Function by Suramin Measured by Rhodamine 123 Retention and Oxygen Consumption in Intact DU145 Prostate Carcinoma Cells

Randall P. Rago, Peter C. Brazy and George Wilding

Cancer Res 1992;52:6953-6955.

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