Lovastatin Augments Apoptosis Induced by Chemotherapeutic Agents in Colon Cancer Cells

b-Hydroxy-b-methylglutaryl coA reductase inhibitors (HRIs) inhibit isoprenylation of several members of the Ras superfamily of proteins and therefore have important cellular effects, including the reduction of proliferation and increasing apoptosis. Significant toxicity at high doses has precluded the use of HRIs as a monotherapy for cancers. We therefore studied whether combinations of the HRI lovastatin with standard chemotherapeutic agents would augment apoptosis in colon cancer cells. In the colon cancer cell lines SW480, HCT116, LoVo, and HT29, lovastatin induced apoptosis with differing sensitivity. Pretreatment with lovastatin significantly increased apoptosis induced by 5-fluorouracil (5-FU) or cisplatin in all four cell lines. Lovastatin treatment resulted in decreased expression of the antiapoptotic protein bcl-2 and increased the expression of the proapoptotic protein bax. The addition of geranylgeranylpyrophospate (10 mM) prevented lovastatin-induced augmentation of 5-FU and cisplatin-induced apoptosis; mevalonate (100 mM) was partially effective, whereas cotreatment with farnesyl pyrophosphate (100 mM) had no effect. These data imply that lovastatin acts by inhibiting geranylgeranylation and not farnesylation of target protein(s). Our data suggest that lovastatin may potentially be combined with 5-FU or cisplatin as chemotherapy for colon cancers. INTRODUCTION Lovastatin is a HRI that reduces cholesterol synthesis by preventing the conversion of b-hydroxy-b-methylglutaryl coA to mevalonate. The formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are downstream of mevalonate in the cholesterol synthetic pathway (1), is also inhibited. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate belong to a class of molecules called isoprenoids that attach to several cellular proteins including G proteins by a posttranslational modification termed isoprenylation. The isoprenylation of G proteins is crucial for membrane attachment and normal functioning (1–4). The low molecular weight G proteins including ras, rho, and rab play crucial roles in signal transduction and therefore influence important cellular functions such as proliferation, apoptosis, and differentiation. By altering the function of these proteins, lovastatin has major effects on cells. HRIs inhibit cellular proliferation and induce apoptosis (5–9), making them potential anticancer agents. However, the use of HRIs in the treatment of cancers, particularly solid tumors, has not been feasible because the doses calculated as required to produce a clinically desirable inhibition of proliferation and increase in apoptosis may be associated with significant toxicity. We postulated that HRIs might augment the apoptosis induced by standard chemotherapeutic agents; thus, they could potentially be added to cancer chemotherapy regimens to improve outcomes. MATERIALS AND METHODS Cell Culture. SW480, HCT116, LoVo, and HT29 cells were obtained from American Type Culture Collection and maintained in DMEM or HAM solution (Life Technologies, Inc., Grand Island, NY) with 10% fetal bovine serum in an atmosphere of 95% O2, 5% CO2 at 37°C without antibiotics. All studies were performed with cells at 50% confluence. Lovastatin was a generous gift from Merck Laboratories (Rahway, NJ). 5-FU and cisplatin were purchased from Sigma (St. Louis, MO). Lovastatin (10 and 30 mM) was used to study the relative sensitivity to lovastatin-induced apoptosis in the four cell lines. Because of markedly different sensitivity to lovastatin-induced apoptosis in the cell lines, the concentrations of lovastatin used in experiments designed to study the effect of combining lovastatin with chemotherapeutic agents were then adjusted to achieve roughly similar levels of apoptosis without 5-FU or cisplatin (SW480 and HCT116 cells, 0, 5, and 10 mM; LoVo and HT29 cells, 0, 10, and 30 mM)]. Because the SW480 and HCT116 cells were more sensitive to 5-FU and cisplatin than LoVo and HT29 cells, we used 50 mg/ml 5-FU and 20 mg/ml cisplatin in studies with SW480 and HCT116 cells and 75 mg/ml 5-FU and 30 mg/ml cisplatin in studies with LoVo and HT29 cells. To determine the relative sensitivity to apoptosis, lovastatin was added to cells at 50% confluence after changing the medium. Apoptosis was quantified after a 48-h incubation by flow cytometry. To study the effect of combinations of lovastatin and 5-FU or cisplatin on apoptosis, lovastatin was added after Received 2/24/99; revised 4/30/99; accepted 5/4/99. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Division of Gastroenterology, Department of Medicine, St. Luke’s-Roosevelt Hospital Center, 1111 Amsterdam Avenue, S&R12, New York, NY 10025. Phone: (212) 523-3680; Fax: (212) 523-3683. 2 The abbreviations used are: HRI, b-hydroxy-b-methylglutraryl coA inhibitor; 5-FU, 5-fluorouracil; TUNEL, terminal deoxynucleotidyl transferase nick end labeling; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; BrdUrd, bromodeoxyuridine. 2223 Vol. 5, 2223–2229, August 1999 Clinical Cancer Research Research. on June 9, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from changing the medium. After a 48-h incubation, the medium was changed again, lovastatin 6 5-FU or cisplatin were added, and the cells were incubated for another 48 h. Apoptosis then was quantified with flow cytometry or MTT assays. For add-back experiments, mevalonate (100 mM), farnesyl pyrophosphate (100 mM), or geranylgeranylpyrophosphate (10 mM; Sigma) were added with lovastatin at the beginning of the experiment and added again at 48 h when the cells were incubated with lovastatin 6 5-FU or cisplatin. A higher dose of lovastatin (30 mM) was used in these experiments to clarify the potential for prevention of the effect of lovastatin. Electron Microscopy. For electron microscopy, adherent and nonadherent (floating) cells were fixed in 1% glutaraldehyde and 4% paraformaldehyde in phosphate buffer, postfixed in 1% osmium tetroxide in phosphate buffer, dehydrated, and embedded in epoxy resin. Ultrathin (80-nm) sections were stained with uranyl and lead acetates and examined with a Zeiss M900 electron microscope at 80 kV. Flow Cytometry. Flow cytometry was used to quantitate apoptotic cells by two methods, DNA histogram and TUNEL staining, which were performed simultaneously on the same samples. TUNEL staining was used to confirm the results observed by measurement of subdiploid cells. TUNEL staining detects cells earlier during apoptosis than measurement of subdiploid cells, so that more cells are determined as apoptotic. Cells were analyzed on a FACSORT flow cytometer (Becton Dickinson, San Jose, CA) after staining using a commercially available Apo-BrdUrd kit (Phoenix Flow Systems, San Diego, CA). TUNEL Staining. DNA strand breaks in apoptotic cells were detected by incorporation of fluorescein-labeled deoxyuridine triphosphate into fragmented DNA by terminal deoxynucleotidyl transferase using the Apo-BrdUrd kit (10). The cells were collected and stained as described by the protocol provided by the manufacturer. The data were plotted on a dot plot, FL2-A versus FL2-W, and a singlet gate was applied. These gated cells were then plotted on dot plot FL1-H(log) versus FL2-A(lin), and cells stained with BrdUrd were counted as apoptotic. The data were also plotted on FL2-H histograms, and the number of subdiploid cells was counted as apoptotic. All flow cytometric studies were performed in triplicate and repeated three times. The data are presented as the mean 6 SD of three readings from each experiment. Similar data were obtained when the experiments were repeated. Student’s t test was done to calculate the statistical significance between the controls (no lovastatin) and the two dose levels of lovastatin used. P , 0.05 was considered significant. MTT Assay. Cells were grown in 96-well plates and treated with lovastatin for 48 h. The medium was then changed, and lovastatin and 5-FU or cisplatin were added. After 48 h, 50 mg of MTT (Sigma) were added to each well, and the plates were incubated at 37°C for 2 h. MTT solubilization solution (10% Triton X-100 and 0.1 N HCl in anhydrous isopropanol; 100 m) was then added, and the plates were agitated on a mechanical shaker to dissolve the crystals. Absorbance was measured spectrophotometrically at a dual wavelength of 570 and 405 nm, and the mean of six readings was used for calculations. The data are presented as the absorbance of treated cells as a percentage of the absorbance of untreated samples. Student’s t test was done to calculate the statistical significance between the controls (no lovastatin) and the two doses of lovastatin used. P , 0.01 was considered significant. Western Blotting. Exponentially growing cells were collected by scraping, washed three times in ice-cold PBS, and resuspended in lysis buffer that contained 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 6 mM mercaptoethanol, 1% NP40, 0.1% SDS, and 10 mM NaF plus the protease inhibitors leupeptin (10 mg/ml) and aprotinin (10 mg/ml), and 0.1 mM phenylethylsulfonyl fluoride (all purchased from Sigma). After lysis with sonification, the resulting insoluble material was removed by centrifugation at 15,000 rpm for 15 min at 4°C and stored at 280°C. Protein concentrations were measured by the Bradford method, and 50-mg samples were mixed with 23 Laemmli buffer, boiled for 5 min, electrophoresed in 10% SDS-PAGE, and transferred to Immobilin membranes (Millipore, Bedford, MA). Western blot analyses were then performed as described previously (11) using specific polyclonal antibodies to Bcl-2 and Bax that were raised to sequence-specific peptides at a concentration of 1:1500 (v/v). Antibody binding was detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL). The images of films were obtained with a digital camera and used for densitometry measurements (KDS 120; Kodak, Rochester, NY).