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Journal of Neuro-Oncology 42: 1–11, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Laboratory Investigation

Lovastatin-induced apoptosis of human medulloblastoma cell lines in vitro Robert J.B. Macaulay1 , Wei Wang1 , Jim Dimitroulakos2 , Lawrence E. Becker2 and Herman Yeger2 Department of Pathology, University of Saskatchewan, Saskatoon, SK, Canada; 2 Department of Paediatric Laboratory Medicine, Hospital for Sick Children, and University of Toronto, Toronto, ON, Canada

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Key words: apoptosis, chemotherapy, human cell lines, lovastatin, medulloblastoma Summary Medulloblastoma is a malignant paediatric central nervous system tumor with a poor prognosis, stimulating the evaluation of improved treatment strategies. Lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is currently used to treat patients with hypercholesterolemia. This compound also inhibits the production of non-steroidal mevalonate derivatives that are implicated in the control of cellular proliferation, and can induce cell-cycle arrest in vitro. We recently showed that lovastatin inhibited growth and promoted apoptosis of neuroblastoma, the peripheral nervous system ‘cousin’ of medulloblastoma. Therefore the potential of lovastatin as a possible anticancer drug against medulloblastoma was evaluated in vitro. Four medulloblastoma cell lines, Daoy, UW228, D341 Med and D283 Med, were treated with 1–40 µM of lovastatin in vitro. Analysis of cell morphologic changes, cell viability, DNA fragmentation and flow cytometry in all four cell lines showed growth inhibition and induction of apoptosis with lovastatin treatment. As little as 10 µM of lovastatin was sufficient to cause a marked reduction in cell numbers, and more than 20 µM of lovastatin induced >90% cells to undergo apoptosis, after intervals ranging between 36 and 96 h, depending on the cell line. Lovastatin induced apoptosis in these cell lines was concomitant with cell cycle arrest in G1. The attached cell lines UW228 and Daoy were more sensitive to lovastatin than D283 Med and D341 Med. Daoy cells which survived several cycles of lovastatin treatment could still be induced to undergo apoptosis after longer treatment times. The efficient induction of apoptosis by lovastatin favours this drug as a potential new avenue of therapeutic intervention for medulloablastoma.

Introduction Medulloblastoma (MB) is the most common central nervous system malignancy of childhood, representing about 20% of childhood intracranial tumors [1] and is a leading cause of cancer-related paediatric mortality. The prognosis of patients with MB is unpredictable, and only about 50% survive after 5 years [2]. Radiation therapy is standard for these patients, but has significant long-term sequelae [3]. The addition or substitution of chemotherapy has met with only limited success [4–6], and dose-limiting toxicity [7], and the development of drug resistance genes has been encountered [8]. Significantly, the mortality of recurrent MB approaches 100% [9], emphasizing the need for new treatment strategies.

The mevalonate pathway, implicated in cellular growth control, is blocked by inhibitors of 3-hydroxy3-methylglutaryl coenzyme A (HMGCoA) reductase, such as lovastatin and its analogues. These compounds have been proposed as potential anticancer drugs in the therapy of human cancer [10,11], and have proved safe and effective as cholesterol-lowing agents [12]. Lovastatin suppresses cholesterol biosynthesis at least partly through inhibition of the synthesis of its precursor mevalonate [13]. In cultured cells, lovastatin induces G1 arrest and cessation of mitotic activity, allowing cell cycle synchronisation by the administration of mevalonate [14,15]. Anti-tumour effects in vitro and in vivo have been reported for lovastatin, alone or in combination with other agents [16–20].

2 We have previously reported that lovastatin induced apoptosis in a number of neuroblastoma (NB) cell lines [16]. The possibility of a similar effect of HMGCoA reductase on MB was considered because of the morphological similarities between MB and NB, and also because of the high levels of HMGCoA reductase expression in the developing cerebellum [16]. We now report that lovastatin induces apoptosis in the medulloblastoma cell lines, Daoy, D283 Med, D341 Med [21] and UW228 [22], confirmed by morphologic, flow cytometric, and DNA electrophoretic evidence. The doses required to induce these phenomena (10– 40 µM) may fall within therapeutically achievable levels if hepatic metabolism is inhibited [23,24], although prolonged therapy at high doses increases the incidence of adverse effects [25]. Thus, lovastatin may represent a potential new therapeutic modality for treatment of MB.

Materials and methods Cell lines MB cell lines Daoy, D283 Med and D341 Med [26] were all obtained from the American Type Culture Collection, and UW228 was a gift from Dr. J.R. Silber [22]. Daoy and UW228 grow as attached flattened cells [27], whereas D283 Med and D341 Med cells only partially attach and grow largely in suspended clusters [28]. These cells were cultured in D-MEM/F12 nutrient mixture supplemented with 10% fetal calf serum, L-glutamine and antibiotics, in a humidified atmosphere of 5% CO2 at 37◦ C. Unless stated otherwise, tissue culture reagents were obtained from Gibco. Lovastatin treatment Cells were grown to subconfluency in flasks, then treated with lovastatin (kindly provided by W.L. Henckler, Merck Research laboratories, Rahway, NJ, USA), or vehicle only, at various times up to 7 days. Conversion of lovastatin from the lactone to the active dihydroxy form was accomplished as described by Keyomarsi et al. [14]. For attached cell lines, media was aspirated and replenished with fresh medium containing varying concentrations of lovastatin. For suspended or partially attached cell lines, floating cells were aspirated, spun and resuspended in medium, then returned to the flasks containing varying concentrations

of lovastatin. For experiments extending beyond 48 h, the medium was removed at this time, floating cells were pelleted by centrifugation, resuspended in fresh medium containing lovastatin at the appropriate concentration, and then replaced into flasks.

Cell viability assays Cell survival was quantified by Trypan Blue exclusion [29]. Briefly, at the indicated time point, control and treated cells were scraped with a rubber policeman. Both adherent and nonadherent cells were isolated by centrifugation, rinsed with PBS, and resuspended. A 100 µl aliquot of this cell suspension was mixed with an equal volume of 0.08% trypan blue in Hank’s balanced salt solution. After 10 min, viable cells were counted by calculating the cells excluding trypan blue with a hemocytometer. Experiments were performed in triplicate, and Student’s paired T -test was applied to the data at maximum time points for all cell lines (Daoy, 48 h; UW228, 40 h; D283 Med and D341 Med, 96 h).

Purification and analysis of DNA Cells were collected as described above, and then lysed with 1.0 ml of digestion buffer (100 mM NaCl, 10 mM Tris-HCl, PH 8.0, 25 mM EDTA, pH 8.0, 0.5% SDS, 0.1 mg/ml proteinase K) and incubated at 50◦ C for 18 h. RNA was digested with RNase A (40 µg/ml) at 37◦ C for 1 h, and DNA extracted with phenol/chloroform, then precipitated with ethanol. DNA representing 2 × 105 cells was electrophoresed in a 1.4% agarose gel containing 0.5 µg/ml ethidium bromide, and photographed under UV light with Polaroid film.

Flow cytometry Flow cytometric analysis was perfomed according to the method of Buchkovich et al. [30]: control and treated cells were collected by centrifugation and washed in ice-cold Hank’s basal salt solution with 0.3 mM EDTA and 0.1% NaN3. Cells were resuspended in 500 µl of the above ice-cold buffer and fixed by the gradual addition of 100% ethanol (−20◦ C) to a final concentration of 80% while vortexing. After 1–3

3 days at 4◦ C, the fixed cells were pelleted and washed once with ice-cold PBS buffer containing 0.3 mM EDTA and 0.1% NaN3, then resuspended in 1 ml of this buffer containing 10 µg/ml boiled RNase A and 5 µg/ml propidium iodide (Sigma). After digestion and staining period at 37◦ C for 30 min, the samples were analyzed by flow cytometry (Coulter(R) Epics(R)). Data were analyzed by overlapped peak multicycle fitting option.

concentration of 1 µM there was no significant effect on morphology of any cell line. Electron microscopic evidence of apoptosis in Daoy and UW228 after 48 h of 20 µM lovastatin treatment was also noted, with fragmentation and condensation of nuclei (data not shown). Thus, despite the differences in morphology between the untreated cell lines, lovastatin-treated cells of each cell line showed similar features indicative of apoptosis. Effect of lovastatin on MB cell viability.

Results Morphological changes in MB cells after lovastatin treatment. At >10 µM lovastatin significant morphological changes were noted (Figure 1). For attached cell lines, cell bodies rounded, and cells became detached from flasks (Figure 1A and B). For the partially attached cell lines, all attachment was lost and cell bodies shrank (Figure 1C and D). Different sensitivities to lovastatin were observed among the cell lines, although all showed dose-dependent effects (Figure 1). Morphological changes were induced in UW228 and Daoy 12– 24 h earlier than for D283 Med and D341 Med. At a

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Treatment of each MB cell line with 10–40 µM lovastatin resulted in significant declines in viable cell number, as assessed by Trypan Blue exclusion. Control untreated cells increased in numbers between 24–96 h (Figure 2); Daoy exhibited a doubling time of approximately, 24 h, while UW228, D283 Med and D341 Med showed doubling times of approximately 36, 48 and 72 h respectively. 1 µM lovastatin had no apparent effect on cell numbers. 10–40 µM lovastatin abrogated the expected increase in viable cell numbers (Figure 2) after 24 h, and thereafter the numbers of viable cells diminished markedly. UW228 and Daoy responded within 24 h, while the responses of D283 Med and D341 Med were

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Figure 1. Morphologic Changes Induced by Lovastatin. The top row shows untreated cells, while the bottom row shows cells following exposure to 20 µM lovastatin, after the stated time interval (phase contrast microscopy). Note extensive cell rounding of attached cell lines, and rounding and fragmentation of cell lines which grow both lightly attached and in suspension. A: Daoy × 48 h. B: UW22 8× 36 h. C: D283 Med × 72 h. D: D341 Med × 96 h.

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Figure 2. Cell Death Induced by Lovastatin. Viability of MB cells was determined by trypan blue exclusion, in untreated cells (0 µM) and after exposure to 1–40 µM lovastatin for up to 96 h. Daoy and UW228 were more sensitive, while D283 Med and D341 Med showed negligible effects until 72 h of treatment.

delayed, with no significant reduction until 72 h. Eventually, all four cell lines showed reduction of viable cell numbers to 90% of Daoy and >80% of UW228 at 24 h, as well as >80% of D283 Med and >90% of D341 Med at 48 h excluded trypan blue, before morphologic changes appeared (Figure 2). To determine whether the treated cells could be rescued after the drug was removed, the treated cells, which were floating, were centrifuged and cultured in fresh media. We found that the fresh media failed to rescue Daoy and UW228 treated for 48 h, D283 Med

5 treated for 84 h, and D341 Med treated for 96 h, consistent with their inability to exclude Trypan Blue. We maintained cultures of the few attached Daoy cells after 48 h of lovastatin treatment, and subjected them to repeated cycles of lovastatin followed by fresh medium. Significantly, 20 µM lovastatin could still inhibit proliferation of these selected ‘resistant’ cells followed by as much as a 90% decline in viable cell number when

treatment was prolonged to 72 h (data not shown; see below and Figures 3E and 4E). Flow cytometric analysis of lovastatin-treated MB cells. To further characterise lovastatin-induced cell death in medulloblastoma cell lines, we analyzed cellular

Figure 3. Flow Cytometric Changes Induced by Lovastatin. Percentages of cells in G0 /G1 , S, and G2 are shown. The left column of graphs represents untreated cells. The right column of graphs represents cells treated with 20 µM lovastatin for the following durations: (A) Daoy, 40 h; (B) UW228, 36 h; (C) D283 Med, 72 h. (D) D341 Med 72 h. (E) Daoy surviving retreated cells, 72 h. The subdiploid shaded peak which appears to the left of the tall control peak (G1) represents the apoptotic fraction.

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Figure 4. DNA Fragmentation Induced by Lovastatin. (A–D) Ethidium bromide-stained agarose gels of medulloblastoma DNA after treatment with varying concentrations of lovastatin. All four cell lines show ‘laddering’ of DNA indicative of apoptosis: (A) Daoy×48 h; (B) UW228 × 40 h; (C) D283 Med × 72 h; (D) D341 Med × 72 h. Lane concentrations: (1) untreated; (2) 1 µM; (3) 10 µM; (4) 20 µM; (5) 40 µM; (M) marker. (E) Surviving retreated cells from Daoy required increased concentrations and longer durations of treatment than naive Daoy cells. Lanes 1–5 and M are as stated above. Lane 6 represents 20 µM × 72 h, and lane 7 is 40 µM × 72 h.

DNA fragmentation using flow cytometry [32]. For each cell line, 20 µM of lovastatin induced the appearance of a subdiploid peak, although at different times for each cell line (Figure 3). This peak, representing nuclear fragmentation due to apoptosis [32], was seen in UW228 and Daoy after 36–40 h of 20 µM lovastatin, while D283 Med and D341 Med required about 72 h treatment. The more rapid appearance of this ‘apoptosis

peak’ in UW228 and Daoy further emphasized their increased sensitivity to lovastatin compared to D283 Med and D341 Med. Treatment with 20 µM of lovastatin was still able to induce apoptosis of surviving ‘resistant’ Daoy cells, but required longer times (72 h) than naive cells (Figure 3E). The size of the apotosis peak increased progressively with increasing dosages between 10 and 40 µM after

7 40 h of treatment (data not shown). Similarly the size of the apoptosis peak resulting from treatment with 20 µM lovastatin increased progressively after first having appeared (data not shown). Lovastatin-induced apoptosis was accompanied by cell cycle arrest in G1 (Figure 3), a phenomenon widely reported in numerous cell cuture systems previously [14,15], although significant cell death has only been observed occasionally [16–20]. Electrophoretic analysis of DNA fragmentation. DNA degradation was evident in all cell lines after treatment with >10 µM of lovastatin (Figure 4). In all four cell lines, nucleosomal oligomers, indicating apoptotic cleavage of chromosomal DNA at internucleosomal loci [33], appeared at variable times correlating with morphological and flow cytometric evidence of apoptosis (Figure 5). Thus, similar to morphologic, viability and flow cytometric changes, DNA ‘laddering’ was dose and time dependent. Control cells cultured equivalent lengths of time without lovastatin failing to demonstrate DNA laddering. DNA ladder formation was more obvious in sensitive cell lines (UW228 and Daoy) compared to less sensitive cell lines (D283 Med and D341 Med). This may relate to the interval between treatment and cell death, since DNA ladder formation in D283 Med was more obvious than in D341 Med, which took 96 h to develop. Rare Daoy cells survived lovastatin treatment and remained attached to the flask. When replenished with fresh medium, such cells resumed proliferation and approached confluence. After several cycles of lovastatin treatment and media replenishment, these cells exhibited reduced sensitivity to lovastatin. Apoptosis could only be induced with 20–40 µM of lovastatin after 72 h of treatment, compared to 36 h for naive cells (Figure 4E).

Discussion Medulloblastoma (MB) arises from neuroepithelial precursors in the developing cerebellum, likely of granule cell lineage [34]. MB recapitulates some phenotypic characteristics of these cells, including high expression of HMGCoA reductase [31]. Although the function of this enzyme in MB has not been clarified, it may well relate to the participation of mevalonate derivatives in cellular proliferation [14] and/or in functional differentiation of neural tissue [16]. Reduction

of mitotic activity in MB cell lines after treatment with lovastatin, an HMGCoA reductase inhibitor [14], is similar to inhibition of mitosis in D283 Med and Daoy upon exposure to phenylacetate; however, no evidence of an apoptotic response was seen with this agent [35]. Apoptosis in the external germinal layer can be induced both in vivo [36] and in primary cultures of fetal cerebellar explants [37]. In addition, some MBs express bcl-2 [38,39], a proto-oncogene which may serve to prevent apoptosis in otherwise susceptible precursors. Thus it is not surprising that MB cells may be induced to undergo apoptosis with appropriate stimulation in vitro [40,41]. We have compiled morphologic, flow cytometric and DNA evidence of extensive apoptosis after exposure of MB cell lines to lovastatin in vitro. The reliance of MB cells on the HMGCoA reductase pathway, as evidenced by high expression of the enzyme [31], may explain the increased sensitivity to inhibition of this enzyme. Initiation of apoptosis in MB cells by lovastatin likely requires mevalonate depletion, the product of the reaction catalyzed by HMGCoA reductase [10]. Preliminary results indicate that inhibition of mevalonate production is a critical step in lovastatin-triggered apoptosis [42]. The three main products of the mevalonate pathway are cholesterol, steroid hormones and non-steroidal derivatives such as farnesyl [10]. Thus, the consequences of mevalonate depletion may be several, any of which may be synergistic, additive or counteractive. The identity of the lovastatin-depleted product of the HMGCoA reductase pathway which suppresses cellular proliferation is unknown; a recent study has narrowed to search to a step proximal to the formation of isopentenyl diphosphate [43]. The proto-oncoprotein p21 ras requires the addition of a farnesyl moiety to allow proper membrane localisation [44–46]. Failure to farnesylate p21 ras results in inability to participate in signal transduction pathways which may participate in cell proliferation, growth and differentiation [11,47]. However, growth inhibitory effects of lovastatin have previously been shown to be independent of farnesylation [44], and may instead be mediated through depletion of other non-steroidal derivatives such as geranylgeraniol [46,48]. Whether specific inhibition of farnesylation will recapitulate the apoptotic effects of lovastatin on MB cell lines is the subject of ongoing investigations. Overexpression of p21 ras has been demonstrated in a minority of MB primary tumors [38] but not MB cell lines; a previous report of [49] N-ras activation in a MB cell line

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Figure 5. Time Course of DNA Laddering Induced by Lovastatin. Ethidium bromide-stained agarose gels of DNA; cells were treated with 20 µM lovastatin after varying intervals. Time intervals for lanes in A (Daoy), B (UW228) and C (D283 Med): (1) 0 h; (2) 12 h; (3) 24 h; (4) 36 h; (5) 48 h; (6) 72 h; (7) Untreated × 72 h. Time intervals for lanes in D (D341 Med): (1) 0 h; (2) 24 h; (3) 48 h; (4) 72 h; (5) 96 h; (6) Untreated × 96 h. (M) marker. Laddering is first apparent at earlier intervals for Daoy and UW228 (36 h) compared to D283 Med (48 h) and D341 Med (72 h).

was invalidated by the subsequent demonstration that the tumour of origin was actually a rhabdomyosarcoma [50]. Effector mechanisms of lovastatin-induced apoptosis have not been established, nor has the relationship of this pheneomenon to other manipulations which may induce apoptosis of MB in vitro [40,41].

The relative resistance of D283 Med and D341 Med to the effects of lovastatin are unexplained, but may be attributable to increased expression of HMGCoA reductase after lovastatin treatment in these cell lines [31]. Alternatively, the differences in morphological phenotype (suspended vs attached), abnormalities of

9 the c-myc gene in D283 Med and D341 Med [28,51] the longer doubling times, or other unknown factors may partially account for these findings. Possible synergy between c-myc and p21 ras overexpression is suggested by recent findings which document cooperation between these two proto-oncogenes in the induction of S-phase [52]. However, the effect of lovastatin on neuroblastoma appears to be independent of the level of N-myc expression [16]. Despite the impressive efficiency of tumour cell death achieved in these experiments, some caution must be exercised before extrapolating these data to patients with MB. First, it is not clear that HMGCoA reductase inhibition alone will be sufficient to achieve significant cell death in vivo; however, tumour xenograft models in immunodeficient mice have been successful in providing evidence of lovastatin efficacy in other tumour systems [20]. Second, the bioavailability of oral lovastatin is limited by the significant firstpass hepatic clearance, and parenteral administration may be necessary to achieve effective CNS dosages [20,53]. Because lovastatin is lipophilic, it is capable of penetrating the blood–brain barrier in concentrations that may have pharmacologic effects [54]. However, even if cell death can be triggered in vivo, required drug dosages in vitro may approach margins of safety [53]. Our observation that Daoy cells retain some sensitivity to lovastatin even after repeated treatment/rescue cycles is encouraging. In addition, other potentiating or differentiating agents may be effective in concert with lovastatin [16,20]. Third, it is not known whether the administration of lovastatin may interfere with the efficacy of conventional anti-neoplastic therapies, if given simultaneously. Typically, such agents depend on DNA replication for their action, and the inhibition of mitosis by lovastatin may abrogate such effects. Finally, permanent cell lines are difficult to establish from MB [55], and those that survive may be biologically different from primary tumours [51]. Although the sensitivity of primary tumours to lovastatin is unknown, the profound tumoricidal effects on all 4 cell lines tested in this study suggests that even those tumours which are capable of surviving in vitro retain sensitivity. In addition, the dismal prognosis attached to recurrent MBs provides a patient population for whom such novel therapies may be the last resort [9]. Ultimately, unravelling the mechanism of lovastatin-induced apoptosis may lead to more effective medical therapy for medulloblastoma, reducing mortality and morbidity following conventional treatment.

Acknowledgements We thank C. McGregor, B. van den Beuken, T. Reichert and J. Alami for their assistance. Operating grants for parts of this work were provided by the Health Services Utilization and Research Commission, Saskatchewan; and by the National Cancer Institute of Canada. RJBM was a Terry Fox Research Fellow, National Cancer Institute of Canada, during part of this study.

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Address for offprints: Rob Macaulay, Department of Pathology, HSB B419, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada; Tel.: (306) 966-6217; Fax: (306) 966-8049; E-mail: [email protected]