Role of ceramide in suramin-induced cancer cell death

ELSEVIER

Cancer Letters 1I9 (1997) I69- I76

Role of ceramide in suramin-induced cancer cell death Jagjit S. Gill*, Anthony J. Windebank Moleculur Neumsciencr Program and Mayo Cancer Center, Mayo Clinic and Mayo Foundation, I.501 Guggmhritn Rrriidinq’. 200 First Street SW, Rochester, MN 55905, USA

Received 12 March 1997; received in revised form 5 May 1997: accepted5 May 1997

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Abstract Suramin is an experimental antineoplastic agent which is currently being tested in clinical trials for its utility in treating breast and prostate cancer. Recent in vitro studies from our laboratory report a disruption in glycolipid metabolism and cell

death in suramin-treated neurons. Evidence presented in this study proposes to consolidate the neurotoxic imndcytotoxic effects of suramin. Electron microscopic studies, his-benzimide staining and DNA gel electrophoresisof suramrn and C?ceramide treatment revealed apoptotic cells in human breast, prostate and rat neuron like cell lines. Apoptotic ceil death was

precededby an elevation in intracellular ceramide. 0 1997 Elsevier Science Ireland Ltd. Keywords:

Breast; Prostate;PC12; Apoptosis; BODIPY ceramide

1. Introduction Suramin, a polysulfonated naphthylurea, was synthesized at the beginning of this century for the treatment of trypanosomiasis and onchocerciasis [l 11. It has since been shown to possess antitumor properties in vitro and in vivo [ 131. Recently reported phase II clinical studies with suramin have demonstrated responses in several human neoplasms including prostate, breast and adrenocortical cancer [4,17,20]. These responses have been observed with prolonged administration or higher doses of the drug. When the plasma levels of suramin exceeded 200 PM (300 &ml), there was, however, an * Correspondingauthor.NeuroscienceResearch,Mayo Clinic and Mayo Foundation, 1.521Guggenheim Building, Rochester, MN 55905, USA. Tel.: +l 507 2841781; fax: +l 507 2843383; e-mail: [email protected]

excessive incidence of significant peripheral neuropathy and coagulopathy. Suramin is a highly charged polyanionic compound which exhibits extensive protein binding. This contributes to its many biological effects, including inhibition of (a) binding of polypeptide growth factors such as platelet derived growth factor. epidermal growth factor, basic fibroblast growth factor. insulin like growth factor-l and transforming growth factor-8 to their cell surface receptors, (b) cell migration and adhesion, (c) angiogenesis, (d) adrenal steroidogenesis, (e) cellular enzymes such as DNA polymerase and topoisomerase, and (f) mitochondrial function. The drug also leads to the accumulation of tissue and circulating glycosaminoglycans [ 131. Which of these, or other yet defined mechanisms. are involved in antitumor effects of suramin are unknown. Earlier studies described an inhibition of growth factor function (i.e. epidermal growth factor, insulin

0304.3835/97/$17.00 0 I997 Elsevier Science Ireland Ltd. All rights reserved PII SO304-3835(97)00272-3

J.S. Gill. A.J. Windebank / Cancer Letters 119 (1997) 169-176

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like growth factor- 1, transforming growth factor-p) associated with the anti-cancer effect of suramin. While suramin was shown to inhibit the growth of breast cancer cell lines in vitro at concentrations achievable in vivo, growth stimulation was seen at lower concentrations [5]. Recent studies in a variety of cancer cells from our laboratory and others have also described a stimulatory effect of suramin on growth factor receptors [5,6,9,15,19,21]. Such a pleiotropic effect on growth factor receptor activation motivated us to consider alternate mechanisms of the chemotherapeutic activity of suramin. In previous in vitro and in vivo studies, we noted that suramin caused the appearance of intracellular lamellar inclusion bodies (LIB) in dorsal root ganglion (DRG) neurons [S]. LIB were morphologically similar to those seen in Tay-Sach’s disease, a lysosoma1 storage disease in which gangliosides accumulate. We have subsequently demonstrated that suramin causes accumulation of the monosialoganglioside GM1 and ceramide in DRG neurons [8]. Suramininduced ceramide accumulation subsequently leads to apoptotic cell death in these DRG neurons. The objective of this study was to investigate the morphological changes in a number of cancer cell lines and to further investigate the cell death process observed in the human breast (MCF7), prostate (DU145) and rat pheochromocytoma (PC12) cell lines exposed to suramin. Morphological changes were compared to those seen in neuronal cells. Measurement of intracellular ceramide and associated apoptotic cell death was performed. Results from this study further support our hypothesis that in both cancer cells and sensory neurons, suramin leads to a lysosomal storage defect, accumulation of ceramide and apoptotic cell death.

2. Materials

sity). All cells were grown on ACLAR (American Chemical, Pleasant Gap, PA) dishes coated with ammoniated and air dried rat tail collagen [3]. Human cancer cells were cultured in RPM1 medium (GIBCO, Gaithersburg, MD) with serum supplementation as described by American Type Culture Collection (Rockville, MD). PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% newborn calf serum and 10% horse serum. Cells were exposed to 100-300 PM suramin for 12 h to 4 days. Cultures were fixed in situ with Trump’s fixative followed by a 1 h postfixation in 1% Os04, stained en bloc with 2% uranyl acetate, dehydrated, and embedded in Spurr’s resin. Cut sections were examined with a Philips CM10 transmission electron microscope. 2.2. Bis-benzimide staining Light microscopic nuclear changes were observed in human breast (MCF7), prostate (DU-145) and PC12 cell lines with the fluorescent DNA stain bisbenzimide (Hoechst 33258; Sigma, St. Louis, MO). Cell cultures were exposed to 100, 200, or 300 PM suramin or 10 PM of the short chain ceramide analog, Czceramide (Matreya, Pleasant Gap, PA) for 12 h to 4 days. Cultures were also exposed to the related lipid analog, dihydroceramide (Matreya), which has not been documented to mediate apoptotic cell death [14]. Following treatment, cell cultures were washed twice in phosphate buffered saline and fixed for a minimum of 1 h in methanol/acetic acid (3:l). Cultures were again washed in phosphate buffered saline and mounted with a drop of 50% glycerol/50% 0.1 M Tris-HCl (pH 7.4) containing 1 pg/ml his-benzimide. Cultures were stored in the dark for a minimum of 15 min and then observed under a fluorescent microscope using Hoechst optics.

and methods 2.3. DNA fragmentation

2.1. Electron microscopy Human cancer cell lines (breastMCF7, colon/ HT29, ovarian/ovcar-3, prostate/DU-145 and rhabdomyosarcoma/AZ04) were generously provided by Dr Matthew M. Ames (Mayo Cancer Center). The rat pheochromocytoma cell line (PC12) was originally obtained from Dr Eric M. Shooter (Stanford Univer-

DNA from cancer cell lines was prepared by modification of a previous method [2]. Cancer cell lines were exposed to the cytocidal concentration of 300 PM suramin. After treatment, cells were washed in phosphate buffered saline and pelleted. Cells were lysed in 200 ~1 of 50 mM Tris-HCl (pH 9.0), 20 mM EDTA, 10 mM NaCl, 1.1% sodium dodecyl sul-

J.S. Gill, A.J. Windebank / Cancer Letter\- II9 (1997) 169.-176

fate (w/v) and 10 mg/ml proteinase K (GIBCO). Cells were incubated in lysis buffer for 48 h at 48°C. Samples were cooled at room temperature for 1.5min and sedimented. Phenol chloroform (200 ~1) was added to each pellet, vortexed, and sedimented again. Following a second phenol chloroform extraction, an aliquot containing 5 pg DNA was added to 50 pg/pl RNase A plus 3 ~1 gel loading buffer and incubated at room temperature for 1 h. Samples were electrophoresed in a 1.2% agarose gel and DNA visualized with ethidium bromide. 2.4. Intracellular

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nelles and the nucleus appeared morphologically sound. After 36-48 h of suramin exposure Seatures characteristic of apoptotic cell death appeared. These included nuclear margination, condensation, cytoplasmic condensation and cell shrinkage. This occurred while nuclear and cellular membrane integrity was retained. Swelling, vacuolation. or membrane disintegration characteristic of necrotic cell death

transport of ceramide

The intracellular fate of ceramide has been studied using an analog in which the fluorophore boron dipyromethene difhmride (ISODIPY) is substituted for the long chain fatty acid side chain. This ceramide analog integrates into the in@aeeIlnlar membranes from exogenous sources and is metabolized similarly to its endogenous sources [l&18]. The use of this BODIPY ceramide compaund (a generous gift from Dr Richard E. Pagano, Mayo Clinic) permitted us to determine the intracellular accumulation of ceramide within a living cell as described previously [16]. Briefly, suramintreated cancer cells were incubated with BODIPYlabeled C,-DMB-ceramide for 30 min at 4“C, washed, and incubated in 10 Helm HEPES-buffered Eagle’s minimum essential me&m (pH 7.4) (without indicator) for 60 tin at 37*C. Lipid extracts were analyzed quantitatively by high pe&ormance thin layer chromatography using 63NdlTfCH@Wl5 mM CaC12(60:35:8 v/v/v) as the devei@ng solvent. The intensity of fluorescent bands were measured by optical densitometry.

3. Results 3. I. Electron microscopy Electron microscopy was carried out in all six cell lines treated with snramin (100-300 PM) or under control conditions. LIB were identified in all cell types treated with suramin but never under control conditions (Fig. 1). LIB appeared within 12 h of suramin exposure. By 24 h, most cells contained many LIB. Within the first 24 h, other cytoplasmic orga-

Fig. 1. Electron micrographs (x3800) of PC12 cells (AI, MW? cells (B) and DU-145 cells (C) exposed to 300 @I sttramin fclr 2 days. In all cells, LIB (arrow) were observed with siiramin cxposure. Suramin treatment also led to the accumulation ol‘ LIB in colonIHT29, ovarian/ovcar-3 and rhabdomyosarcoma/A%o4 ceil lines(data not shown). Cells with fragmented and condensed nuclei (*), characteristic of apoptotic cell death, were observt~d in all srll lines exposed to suramin.

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were only observed with prolonged suramin treatment (4 days). 3.2. Bis-benzimide staining Human prostate (DU-145), breast (MCF7) cancer cells and rat PC 12 cells exposed to suramin were used to observe light microscopic nuclear changes consistent with apoptotic cell death. Bis-benzimide staining of cultures treated with 100-300 PM suramin revealed fragmented and condensed nuclei by 48 h

(Fig. 2). These apoptotic nuclei correlated with condensed cells under phase contrast microscopy. Cells treated with suramin retained the integrity of their plasma membrane. The cancer cell lines were also exposed to the cell permeable short chain ceramide analog (C2-ceramide; 10 PM) for 12-48 h (Fig. 2). C2-ceramide treatment also induced nuclear blebbing and condensation. Apoptotic cells were observed by 24 h C2-ceramide treatment. Cultures exposed to the Cz-ceramide analog, dihydroceramide (10 PM), did not display any

Fig. 2. Human prostate cancer cells (DU-145) were exposed to 300 PM suramin (C,D) or 10 1M C,-ceramide (E,F) for 2 days, fixed and stained with his-benzimide (Hoechst 33258). Phase (A,C,E) and fluorescence (B,D,F) microscope images were obtained from representative areas. Condensed and fragmented nuclei (arrow) were observed with his-benzimide staining in cells treated with suramin and Cs-ceramide. Apoptotic cells were not observed in untreated/control cells (magnification x400). Apoptotic cells were also observed in breast/MCF7 and PC12 cells exposed to suramin and C,-ceramide.

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ted with 300 PM suramin for 24 h followed by incubation with the fluorescent ceramide analog, BODIPY ceramide. Isolation and subsequent separation of cellular glycolipids revealed a significant accumulation of ceramide in all suramin-treated cancer cells as compared to untreated cultures (Fig. 4). Optical densitometry revealed 3.66 (kO.45),4.1 (k0.63) and 2.82 (+-0.5 1) fold increases in ceramide levels in suramintreated MCF7, DU-145 and PC12 cells, respectively. Significant ceramide accumulation (24 h) in suramintreated cultures was observed before apoptotic cell death as described with his-benzimide staining or DNA laddering (48 h). lUW7

Pr estate (DU- 145)

Breast (MCF7)

DIJ-146

PC12

B

Fig. 3. Human prostate (DU- 145) and breast (MCF7) cancer cells were exposed to 300 pM suramin (Sur) for 48 h. Isolated DNA was separated on a 1.2% agarose gel and visualized by ethidium bromide. DNA laddering was observed in suramin-treated cultures while control (Ctl) cultures did not display any laddering.

cells with fragmented or condensed nuclei (data not shown). Cultures treated with dihydroceramide resembled those cultured under control conditions. 3.3. DNA ,fragmentation DNA was isolated from cancer cell lines exposed to 300 PM suramin (48 h) and separated by gel electrophoresis on a 1.2% agarose gel (Fig. 3). In all three cell lines studied (MCF7, DU-145 and PC12), internucleosomal DNA fragmentation was observed in suramin-treated cultures. DNA laddering was not detected in untreated cancer cell lines. 3.4. Suramin-induced ceramide accumulation To determine the role of intracellular ceramide accumulation and suramin-induced apoptotic cell death, MCF7, DU-145 and PC12 cell lines were trea-

MCF7

DU-145

PC12

Fig. 4. (A) Cancer cell lines (breast (MCF7), prostate (DU- 145) and pheochromocytoma (PC12)) were exposed to 300 &l summin for 24 h and incubated with the BODIPY-labeled ceramide analag, C5DMB-ceramide. Lipid extracts were analyzed quantitatively by high performance thin layer chromatography. Fluorescent lipids were visualized. Standards (Cer, ceramide; GluCer, glucosylceramidej are presented in the flanking lanes. Suramin treatment (+) revealed a significant accumulation of intracellular ceramide as compared to untreated cultures (-). (Bj Optical densitometry from high performance thin layer chromatography plates described in (A) revealed a significant fold increase in BGDIPY-labeled ceramide in all three suramin-treated cancer cell lines. Bars reflect the mean f SEM of three high performance thin layer chromatography measurements.

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4. Discussion The mechanism by which suramin mediates its antitumor effects on cancer cells needs to be clarified. As a highly charged polysulfonated naphthylurea, suramin is able to bind a number of proteins including growth factors and/or their receptors. Although inhibition of growth factors provided the initial rationale for testing suramin in breast and prostate cancer, it is not certain that this mechanism mediates the antitumor effect. Studies presented here suggest that the antitumor effects of suramin relate to a ceramide mediated cell death pathway. In this paper, the effect of suramin on intracellular ceramide and its relation to apoptotic cell death in human breast, prostate and rat pheochromocytoma cell lines is discussed. Electron micrographs revealed the accumulation of LIB in cancer cell lines exposed to suramin. The morphology and temporal accumulation of these LIB were similar to those observed in suramin-treated DRG neurons [8]. Cancer cell lines exposed to suramin displayed internucleosomal fragmentation. Light microscopic analysis of MCF7, DU145 and PC12 cells revealed the induction of apoptotic cell death by both a cell permeable ceramide analog (CZ-ceramide) and suramin at 24 and 48 h, respectively. Analysis of intracellular ceramide revealed a significant accumulation of the fluorescent ceramide analog, BODIPY ceramide, in suramin-treated cancer cells before apoptotic cell death. In all six cancer cell lines studied, suramin treatment resulted in nuclear condensation and fragmentation (Fig. 1). Cells with condensed nuclei also displayed cell shrinkage while retaining the integrity of both the nuclear membrane and plasma membrane. Such morphological changes are characteristic of apoptotic cell death. Organelle swelling and cell lysis was observed only at 4 days suramin treatment, suggestive of a necrotic cell death process. Similar descriptions of necrotic cell death preceded by apoptosis have been reported previously with growth factor withdrawal and after continuous exposure to toxic agents [ 1,23,24]. Nuclear changes characteristic of apoptotic cell death in cancer cell lines exposed to suramin were also observed at the light microscope level (Fig. 2). The fluorescent nuclear stain his-benzimide revealed nuclear condensation and blebbing in cancer cells

treated with suramin. Phase contrast microscopy revealed an intact plasma membrane in cells with fragmented or condensed nuclei. Additional evidence for an apoptotic cell death process was demonstrated by DNA laddering, characteristic of intemucleosomal fragmentation, in suramin-treated cancer cells (Fig. 3). Collectively, the morphological changes and disintegration of DNA integrity as observed by gel electrophoresis suggests that suramin induces an apoptotic cell death process in the cancer cells studied. Morphological analysis also provided us with insights into the mechanism of suramin-induced apoptotic cell death. In all cancer cell lines studied, suramin treatment led to the accumulation of LIB (Fig. 1). These LIB appeared quickly, within 12 h of suramin exposure, and dying cells were full of LIB. LIB accumulation was found to precede cancer cell death. The morphology and temporal accumulation of these LIB were similar to those observed in suramin-treated DRG neurons [7,8]. These previous studies demonstrated a suramin-induced accumulation of the monosialoganglioside GM, and ceramide which colocalized to the LIB. This led us to consider the effect of suramin on glycolipid metabolism in cancer cells and its role in cell viability. Ceramide trafficking studies allowed us to evaluate the intracellular accumulation of ceramide in suramin-treated cancer cells (Fig. 4). In the human breast, prostate and rat pheochromocytoma cell lines examined, there was a significant accumulation of the ceramide analog, BODIPY ceramide, with suramin treatment as compared to control cultures. This accumulation was measured at 24 h, a time point before any apoptotic cell death could be observed. This association would indicate that ceramide accumulation is not a consequence of drug-induced cell death but rather is involved in the cell death signal cascade propagated by suramin. Evidence to further support a ceramide mediated cell death process associated with suramin treatment was demonstrated by the nuclear condensation and fragmentation observed in cancer cells exposed to (&ceramide (Fig. 2). This cell permeable short chain ceramide analog was able to cause nuclear blebbing and condensation in all three cancer cell lines tested, while maintaining the integrity of the plasma membrane of these cells. The induction of apoptotic cell death by Cz-ceramide was specific, as the cera-

J.S. Gill, A.J. Windebank/Cancer Letters 119 (1997) 169-176

mide analog dihydroceramide was inactive in inducing nuclear fragmentation or condensation. The onset of ceramide mediated apoptosis (12 h) in the cancer cells described in this study was comparable to previous reports in other cancer cell lines [lo, 12,141. This time course is compatible with the activation of a regulated program involving new gene transcription and/or protein synthesis. The longer period associated with suramin mediated changes in nuclear integrity (48 h) would correspond to a disruption in glycolipid/ganglioside metabolism as evidenced by LIB formation with secondary accumulation of ceramide levels sufficient to trigger a ceramide mediated cell death cascade. Results from this study demonstrate that the cytotoxic effect of suramin in cancer cells may involve abnormalities in glycosphingolipid metabolism and ceramide mediated apoptotic cell death. Recent studies from our laboratory describe a similar disruption of glycolipid metabolism in suramin-treated DRG neurons with secondary accumulation of ceramide and subsequent apoptotic cell death (unpublished data). A link between rapidly dividing cancer cells and postmitotic sensory neurons is their relatively high rates of glycosphingolipid (ganglioside) synthesis. Thus, we propose that the neurotoxic insult and chemotherapeutic effects of suramin may share similar cellular pathways, namely the activation of a ceramide mediated programmed cell death signal cascade. A recent study by Tu et al. [22] determined that suramin-induced cell death in the Dunning-G prostate carcinoma cell line was not substantially influenced by the expression of bcl-2, a mediator of apoptotic cell death. This finding implies that suramin may be activating a cell death pathway independent of bcl-2 or, alternatively, may result in alterations downstream of hcl-2 regulation. Although bcl-2 has not been associated with ceramide mediated apoptotic cell death, defined substrates in the ceramide mediated cascade have yet to be fully described. Determination of these elements will be the focus of future studies.

Acknowledgements The excellent secretarial assistance of MS Linda A. Goldbeck is greatly appreciated. We thank Dr Richard

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E. Pagan0 for his technical assistance in the ceramide trafficking studies.

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