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. 2003 Aug 26;129(11):631–641. doi: 10.1007/s00432-003-0490-2

Epidermal growth factor receptor-targeted therapy potentiates lovastatin-induced apoptosis in head and neck squamous cell carcinoma cells

Angela J Mantha 1, Kathryn E McFee 1, Nima Niknejad 1, Glenwood Goss 1, Ian A Lorimer 1, Jim Dimitroulakos 1,✉
PMCID: PMC12161920  PMID: 12942316

Abstract

Purpose

Mevalonate metabolites are vital for a variety of key cellular functions with the biosynthetic products including cholesterol and farnesyl and geranylgeranyl isoprenoids. Inhibition of this pathway using lovastatin induces a potent apoptotic response in a specific subset of human tumor-derived cell lines, including head and neck squamous cell carcinomas (HNSCC). In this study, we evaluated the potential of a number of chemotherapeutics that demonstrate activity in HNSCC, including an inhibitor of epidermal growth factor receptor (EGFR) to potentiate the cytotoxic effects of lovastatin.

Methods

We evaluated the cytotoxic effects of combining a variety of chemotherapeutics with lovastatin using the MTT assay and flow cytometry. The MCF-7 lovastatin-resistant breast adenocarcinoma cell line and the lovastatin-sensitive HNSCC cell lines SCC9 and SCC25 were tested. Expression levels of EGFR and ligand activated EGFR following lovastatin treatment were analyzed by Western blotting.

Results

Pretreatment or concomitant treatment of 10 μM lovastatin did not significantly augment the effects of a variety of chemotherapeutic agents tested in these cell lines. Co-administration with actinomycin D or cycloheximide, drugs that inhibit RNA and protein synthesis, respectively, inhibited lovastatin-induced apoptosis in these cell lines. This suggests a requirement for the cellular functions disrupted by these chemotherapeutic agents in lovastatin-induced apoptosis of HNSCC cells. In contrast to the chemotherapeutics analyzed, the AG1478 tyrosine kinase inhibitor of the EGFR demonstrated additive cytotoxic effects in combination with lovastatin in HNSCC cells. Mevalonate metabolites may regulate EGFR function, suggesting that lovastatin may inhibit the activity of this receptor. Indeed, lovastatin treatment inhibited EGF-induced autophosphorylation of the EGFR in the SCC9 and SCC25 cell lines. Pretreatment of SCC9 and SCC25 cell lines for 24 h with 10 μM lovastatin, conditions that demonstrated significant inhibition of EGF-induced EGFR autophosphorylation, induced significant additive effects in combination with AG1478.

Conclusion

These results demonstrated the ability of EGFR pathway inhibitors to potentiate lovastatin-induced apoptosis and suggested that lovastatin may target the EGFR pathway in HNSCC cells.

Keywords: Experimental therapeutics, Mevalonate pathway, HMG-CoA reductase, Epidermal growth factor receptor

Introduction

HNSCCs include epithelial malignancies of the oral cavity, pharynx and larynx (Vokes et al. 1993). As a group, they represent the sixth most common human neoplasm, with an estimated annual worldwide incidence of 500,000 cases (Boring et al. 1992; Vokes et al. 1993). Despite significant advances in treatment using recent protocols for surgery, radiation and chemotherapy, the long-term survival of HNSCC patients has remained approximately 50% for the last 3 decades (Atula et al. 1997; Boring et al. 1994). Identification of active drugs in HNSCC and the optimization of scheduling and combination therapy has been a central component of these therapeutic approaches. Historically, the most active single agent in metastatic or recurrent HNSCC is cisplatin, which produces a response rate of up to 30% and an increase in median survival from 4 to 6 months in these patients (Pinto and Jacobs 1991; Wittes et al. 1977). Cisplatin is a platinum-based compound that causes cytotoxicity through its ability to form non-repairable adducts in DNA (Cohen and Lippard 2001) that trigger apoptosis (Trimmer and Essigmann 1999). Other agents that have demonstrated activity as single agents in HNSCC include 5-fluorouracil (5-FU) and paclitaxel (Forastiere et al. 1998; Pinto and Jacobs 1991). Their mechanisms of actions differ from cisplatin and possess response rates of less than 30% (Forastiere et al. 1998; Pinto and Jacobs 1991). 5-FU is a uracil analogue that is extensively incorporated into RNA, affecting function, and also inhibits the formation of thymidine, resulting in the inhibition of DNA synthesis (Grem 2000). Paclitaxel binds to and stabilizes microtubules, inducing a well-defined G2-M arrest (Dumontet and Sikic 1999). In addition, paclitaxel blocks mitosis at the metaphase/anaphase transition because of interference with spindle formation (Dumontet and Sikic 1999). This mitotic block is considered to be the primary mechanism for the cytotoxic effects of paclitaxel resulting in the induction of apoptosis.

This relatively weak response rate of HNSCC to traditional chemotherapeutics is thought to stem from the ability of these tumor cells to acquire resistance to these agents as well as the inherent toxicities associated with these treatments, particularly in this patient population (Lamont and Vokes 2001). The incorporation of novel agents in treatment regimens that do not exhibit significant toxicities and target tumor cells specifically, therefore, may have clinical benefit. To this end, we demonstrated that targeting 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate pathway, results in tumor-specific cytotoxicity through the induction of a potent apoptotic response, particularly in HNSCC-derived cell lines (Dimitroulakos et al. 1999; Dimitroulakos et al. 2001; Dimitroulakos and Yeger 1996). The diverse array of critical biosynthetic products of the mevalonate pathway include sterols, especially cholesterol, involved in membrane structure and steroid production; ubiquinone, involved in electron transport; farnesyl and geranylgeranyl isoprenoids whose covalent binding to proteins such as ras facilitates their membrane localization; dolichol, required for glycoprotein synthesis; and retinoic acid precursors (Goldstein and Brown 1990).

Lovastatin is a specific, non-reversible competitive inhibitor of HMG-CoA reductase (Corsini et al. 1995; Hunninghake 1992), whose ability to block this critical metabolic pathway has led to its extensive clinical use as a treatment for hypercholesterolemia (Corsini et al. 1995; Hunninghake 1992). Targeting HMG-CoA reductase using lovastatin induces a potent apoptotic response in HNSCC-derived cell lines at therapeutically achievable levels of this drug (Dimitroulakos et al. 1999; Dimitroulakos et al. 2001; Dimitroulakos and Yeger 1996). Based on these data, a phase I trial evaluating the potential of lovastatin in the treatment of recurrent metastatic HNSCC was undertaken by our group (Princess Margaret Hospital, Toronto, Canada). Although no tumor regressions were documented, lovastatin demonstrated the ability to induce disease stabilization (4/11 patients) in this cohort (Knox, J.J., et al. manuscript in preparation). The most relevant clinical application of lovastatin in HNSCC would likely be as a part of a combined modality approach. Along these lines, a phase I trial using pravastatin, another member of the statin family of HMG-CoA reductase inhibitors, in combination with 5-FU demonstrated enhanced overall survival compared to 5-FU treatment alone in hepatoma patients (Kawata et al. 2001). Furthermore, a number of studies have demonstrated the potential of lovastatin to modestly potentiate the cytotoxic effects of chemotherapeutics in colon cancer and melanomas (Agarwal et al. 1999; Feleszko and Jakobisiak 2000; Feleszko et al. 2000; Feleszko et al. 2002). To date, similar combination chemotherapy studies in HNSCC that are particularly sensitive to the apoptotic effects of lovastatin have not been performed. In this study, we evaluated in vitro efficacy of combining standard chemotherapeutic agents with lovastatin in HNSCC. We included agents with various modes of action and focused on chemotherapeutics that have demonstrated activity in HNSCC.

Another potential therapeutic approach in HNSCC has revolved around targeting of the epidermal growth factor receptor (EGFR) (Mendelsohn and Baselga 2000). EGFR is over-expressed in the majority of HNSCC and when stimulated by binding to its ligands is a potent stimulator of their growth and survival (Hoffmann et al. 1997; van Gog et al. 1998). Blocking antibodies that inhibit ligand binding exert several biological activities in a number of in vitro and in vivo models, including modulation of tumor cell proliferation, differentiation and apoptosis (Hoffmann et al. 1997; van Gog et al. 1998). Antibody directed therapy against EGFR, however, has demonstrated only limited efficacy in the treatment of minimal residual disease (Hoffmann et al. 1997; van Gog et al. 1998). More recently, specific EGFR selective tyrosine kinase inhibitors have been developed to target the function of this receptor by inhibiting the transduction of its mitotic and survival signals (Arteaga and Johnson 2001; Mendelsohn and Baselga 2000). These kinase inhibitors have demonstrated significant effects on HNSCC growth and survival in vitro and are currently being evaluated in clinical trials (Herbst 2002).

Mevalonate metabolites play an essential role in transducing EGFR-mediated signaling. Proteins that require farnesyl or geranylgeranyl for their membrane localization and activity are downstream signaling mediators of EGFR including the ras, rho and rab family of proteins (Pruitt and Der 2001; Takai et al. 2001). Dolichol is a molecular chaperone that is involved in the N-linked glycosylation of a number of receptor tyrosine kinases, including EGFR, which facilitates their proper conformation and localization (Bishayee 2000; Slieker et al. 1988). Because of the potential of lovastatin to affect EGFR signaling through its ability to target HMG-CoA reductase, we also evaluated the effect of an EGFR tyrosine kinase inhibitor on lovastatin-induced apoptosis of HNSCC cells.

Materials and methods

Tissue culture

The SCC9 and SCC25 HNSCC and the MCF-7 breast adenocarcinoma cell lines were obtained from the ATCC (Rockville, Md.). The cell lines were maintained in Dulbeco's-MEM (Media Services, Ottawa Regional Cancer Centre) supplemented with 10% fetal bovine serum (Medicorp, Montreal). Cells were exposed to solvent control or to 0–100 μM lovastatin [generously provided by Apotex, Mississauga, Canada, diluted from a 10 mM stock in ethanol prepared as previously described, (Dimitroulakos and Yeger 1996)]. AG1478 (Calbiochem, San Diego) was diluted from a 50-mM stock in DMSO and human recombinant EGF (Sigma) was diluted from a 50-μg/ml stock in 10 mM acetic acid/0.1% bovine serum albumin (Sigma). The chemotherapeutics were obtained from the pharmacy at the Ottawa Regional Cancer Centre.

MTT assay

In a 96-well flat bottom plate (Nunc, Naperville, Ill.) approximately 5,000 cells/150 µl of cell suspension was used to seed each well. The cells were incubated overnight to allow for cell attachment and recovery. Following treatment, 50 µl of a 5 mg/ml solution in phosphate buffered saline of the MTT tetrazolium substrate (Sigma) was added and incubated for up to 6 h at 37°C. The resulting violet formazan precipitate was solubilized by the addition of 100 µl of a 0.01 M HCl/10% SDS (Sigma) solution shaking overnight at 37°C. The plates were then analyzed on an MRX Microplate Reader from Dynex Technologies at 570 nm to determine the optical density of the samples.

Flow cytometry

Cell cycle parameters were determined by flow cytometry using propidium iodide labeling of single cells as described previously (Dimitroulakos et al. 2001). Single cell suspensions were labeled with 50 µg/ml propidium iodide (Sigma) and approximately 106 cells in 100 µl analyzed by flow cytometry. Ten thousand cells were evaluated, and the percentage of cells in subG1 phase was determined using the Modfit LT program (Verity Software House, Topsham, Mass.).

Western blot analysis

Total cellular protein was extracted using a buffer that consisted of 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate (Sigma), 0.1% SDS (Sigma), 0.2 mM sodium orthovanadate (Sigma) and 0.2 mM phenyl methyl sulphonyl fluoride (Sigma) in 2×PBS. Approximately 200 μl of extraction buffer was used to treat 106 cells. Total protein was quantified with the BioRad Protein Assay using bovine serum albumin (Sigma) as standard. Protein extracts representing 20 μg total protein from the cell lines and their treatments were separated on a 10% SDS-PAGE gel and electrophoretically transferred onto PVDF membranes (Amersham, Toronto). Membranes were blocked in 5% skim milk powder in PBS overnight at 4°C. Primary antibody, diluted in 5% skim milk powder in PBS, was incubated with the membrane for 1 h at room temperature. The polyclonal antibodies specific for EGFR and phospho-EGFR at site 1068 (Cell Signaling Technology, Beverly, Mass.) were used. The secondary antibody (Amersham) was applied at a 1:5,000 dilution in 5% skim milk powder in PBS and incubated for 1 h at room temperature (washes following antibody incubations are 3×5 min in PBS/0.05% Tween 80 (Sigma) then processed for chemiluminescent detection (Amersham). After the desired exposure was obtained the membrane was stained with Coomassie Blue (Sigma) to ensure equal loading of the samples.

Results

In this study, we focused on the potential of lovastatin to augment the cytotoxic effects of chemotherapeutic agents that have shown clinical activity in HNSCC. Due to the significant effects of cisplatin in HNSCC, we also included carboplatin and oxaliplatin in this study as they represent structurally distinct second and third generation platinum compounds, respectively (Kelland and McKeage 1994; Raymond et al. 1998). We compared the effects of combining cisplatin, 5-FU, paclitaxel, carboplatin and oxaliplatin in the HNSCC cell lines SCC9 and SCC25 as well as the breast adenocarcinoma cell line MCF-7 as a comparator. These cell lines represent the spectrum of sensitivity to lovastatin-induced cytotoxicity as MCF-7 cells are resistant, SCC9 display an intermediate response while SCC25 are sensitive to the apoptotic effects of this agent (Dimitroulakos et al. 1999) (Fig. 1A). The use of these three cell lines allowed the evaluation of the potential of lovastatin to augment the effects of traditional chemotherapeutics in cells that show differential sensitivities to lovastatin.

Fig. 1.

Fig. 1.

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Evaluating the effects of lovastatin on the viability of the MCF-7, SCC9 and SCC25 tumor-derived cell lines using MTT assay and flow cytometric analysis. A MTT enzyme activity after exposure to 0–100 μM lovastatin for 48 h, highlighted the three responses observed with this agent; non-responsive (MCF-7), intermediate (SCC9) and sensitive (SCC25). B Representative flow cytometric analyses of MCF-7 and SCC25 cell lines after exposure to solvent control, 10 μM or 50 μM lovastatin for 48 h. The percentage of cells in the subG1 (apoptotic) fraction is shown in the upper left region and the percentage of cells in each cell cycle phase is in the upper right region of each histogram. SCC25 cells displayed dramatic cell cycle and apoptotic responses to lovastatin exposure compared to MCF-7 cells

Flow cytometric analysis confirmed that this differential sensitivity was a result of significant growth inhibitory and apoptotic responses triggered in the sensitive SCC25 cell line that was not evident in MCF-7 cells under these experimental conditions (Fig. 1B). Cell cycle distribution and induction of apoptosis were visualized and quantified by determining cellular DNA content using propidium iodide staining (Darzynkiewicz et al. 1992; Piacentini et al. 1993). Apoptosis generally results in cellular and nuclear fragmentation with the formation of apoptotic bodies resulting in a subG1 population of cells (Darzynkiewicz et al. 1992; Piacentini et al. 1993). The S-phase fraction of SCC25 cells in cycle was dramatically reduced by lovastatin from 23.9% in untreated cells to 5.3% in cells treated with 10 μM for 48 h with a significant G1 cell cycle arrest from 48.8 to 68.4% in 10 μM 48-h treated cells (Fig. 1B). In this study, MCF-7 cells showed minimal cell cycle effects with this drug under these conditions. In the SCC25 cell line, lovastatin treatment also showed a significant percentage of cells in the apoptotic subG1 fraction that was not evident in the MCF-7 cell line. Lovastatin-induced apoptosis was significant at both 10 μM (20.4%) and 50 μM (34.0%) 48-h treatments (Fig. 1B). The SCC9 cell line is composed of a mixture of diploid and tetraploid cells and, although evident, evaluations of subG1 fractions due the complexity of the histogram were difficult to assess quantitatively using this method (data not shown).

Lovastatin does not significantly potentiate the cytotoxic effects of chemotherapeutics in HNSCC cells

To determine whether lovastatin can enhance the cytotoxicity induced by chemotherapeutic agents in HNSCC cells, we treated the MCF-7, SCC9 and SCC25 cell lines with 10 μM lovastatin in combination with a variety of standard chemotherapeutic agents. The concentration of lovastatin used is pharmacologically relevant as phase I clinical trials have demonstrated achievable serum concentrations of up to 4 μM sustainable over 7 days in cancer patients (Thibault et al. 1996). Various concentrations of cisplatin, 5-FU, paclitaxel, carboplatin and oxaliplatin with or without the addition of lovastatin were evaluated by the MTT cytotoxicity assay following 48-h treatment (Fig. 2). The concentrations of these drugs and the time-point used was based on the NCI 60 cell line screen of these agents that assessed their sensitivity in a wide panel of human tumor-derived cell lines (Koutsoukos et al. 1994; Monks et al. 1997). In the MCF-7 cell line, lovastatin did not potentiate the cytotoxic effects of the drugs tested. There were no significant differences in cytotoxic effects when evaluating both lovastatin alone and drug alone versus their combination in all of the concentrations of drugs used (paired t-test analysis, data not shown). In the SCC9 cell line that shows intermediate sensitivity to lovastatin, borderline significant differences in both the lovastatin alone and drug alone versus their combination was limited to a single low dose of paclitaxel and oxaliplatin (paired t-test, data not shown). The SCC25 cell line that demonstrates sensitivity to lovastatin-induced apoptosis, did not demonstrate any significant differences in cytotoxicity when evaluating both lovastatin alone and drug alone versus their combination (paired t-test analysis, data not shown).

Fig. 2.

Fig. 2.

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Evaluating the effects on cell viability of combining 10 μM lovastatin with a variety of chemotherapeutics agents including cisplatin, 5-fluorouracil, paclitaxel, carboplatin and oxaliplatin. Cell viability was determined by MTT analysis following 48-h treatment with these combinations of agents. A variety of concentrations of each chemotherapeutic were analyzed alone or in combination with 10 μM lovastatin. No significant differences in responses were evident between either lovastatin and chemotherapeutic treatments alone and their combinations (paired t-test, data not shown)

To evaluate this lack of co-operativity, the cell cycle and apoptotic effects of cisplatin and 5-FU ± lovastatin treatments for 48 h in the SCC25 cell line were analyzed by flow cytometric analysis. Based on their cytotoxic profiles determined by MTT analysis, both low and high concentrations of cisplatin (1 and 5 μg/ml) and 5-FU (100 and 1,000 μM) ±10 μM lovastatin were evaluated (Fig. 3). Cisplatin induced a pronounced S-phase, while 5-FU induced a potent G2/M cell cycle arrest as has been previously reported for these agents (Cohen and Lippard 2001; Grem 2000). At the low concentrations of these agents, lovastatin-induced apoptosis was evident as prominent subG1 population of cells; however, the overall cytotoxicity as determined by MTT analysis is not enhanced by these combinations as the apoptotic fraction of cells remains at the levels of lovastatin treatment alone. At the high doses of the chemotherapeutics, lovastatin-induced apoptosis was inhibited and showed no augmentation of the apoptosis induced by cisplatin or 5-FU alone (Fig. 3).

Fig. 3.

Fig. 3.

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Flow cytometric analysis of cisplatin and 5-FU-treated SCC25 cells alone or in combination with 10 μM lovastatin for 48 h. The subG1 (apoptotic) fraction of cells is displayed in the upper left region of the individual histograms. Combinations of either high or low concentrations of cisplatin or 5-FU did not potentiate the apoptotic effects of 10 μM lovastatin in SCC25 cells

In colon carcinoma cells, a recent report demonstrated that pretreatment with lovastatin for 48 h followed by combinations with cisplatin and 5-FU for an additional 48 h augmented lovastatin-induced apoptosis (Agarwal et al. 1999). These results were not additive but were more pronounced at concentrations of these chemotherapeutics that displayed weak cytotoxic responses (Agarwal et al. 1999). Similarly, pretreatment of MCF-7 and SCC9 with 10 μM lovastatin for 48 h followed by the combination with low dose cisplatin (0.1 and 1 μg/ml) for 48 h showed a weak augmentation of the cytotoxic effects of lovastatin (Fig. 4). No significant effects were demonstrated with lovastatin pretreatment in combination with 5-FU in these cell lines. The lovastatin-sensitive cell line SCC25, pretreatment with lovastatin followed by co-administration of cisplatin or 5-FU failed to demonstrate co-operativity (Fig. 4).

Fig. 4.

Fig. 4.

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Evaluating the cytotoxic effects of a 48-h pretreatment of 10 μM lovastatin in combination with cisplatin and 5-fluorouracil. MCF-7, SCC9 and SCC25 cells were pretreated with solvent control or lovastatin followed by 48-h treatment with cisplatin or 5-flourouracil and cell viability determined by MTT assay. The lovastatin pretreatment was maintained through replenishment after the duration of the pretreatment. No significant additive effects with these chemotherapeutics were demonstrated in these cell lines with pretreatment with lovastatin

Actinomycin D and cycloheximide inhibit lovastatin-induced apoptosis

The lack of significant co-operativity between lovastatin and the chemotherapeutics tested in HNSCC cells suggests that the cellular functions disrupted by the cytotoxic doses of these chemotherapeutics may be required for lovastatin-induced apoptosis. In this study, we evaluated the requirement of transcription and translation in the apoptotic responses induced by lovastatin in HNSCC. Actinomycin D and cycloheximide are potent inhibitors of transcription and translation, respectively (Kim et al. 1998). Both agents have been used extensively to evaluate the roles of transcription and translation on biological processes. The MCF-7, SCC9 and SCC25 cell lines were treated with various concentrations of lovastatin with or without 5 ng/ml actinomycin D or 0.1 μM cycloheximide for 48 h and evaluated by MTT analysis (Fig. 5A). These concentrations of actinomycin D and cycloheximide have been shown to inhibit transcription and translation in a number of cell types including HNSCC cells (Kim et al. 1998). As expected, both of these drugs significantly inhibited MTT activity with actinomycin D demonstrating a weak apoptotic response (Fig. 5). In the MCF-7 and SCC9 cell lines, MTT activity of actinomycin D and cycloheximide remained relatively unchanged even with the addition of up to 50 μM lovastatin, indicating that these drugs inhibited the effects of lovastatin on these cells (Fig. 5A). In the lovastatin-sensitive SCC25 cell line, actinomycin D and cycloheximide protected against lovastatin-induced cytotoxicity as overall cell viability was increased in the combinations of agents as compared to lovastatin treatment alone. Flow cytometric analysis of these combinations in SCC25 cells showed an inhibition of lovastatin-induced apoptosis (Fig. 5B). These results suggest a requirement for these cellular functions to facilitate lovastatin-induced apoptosis.

Fig. 5.

Fig. 5.

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Evaluating the effects of actinomycin D (ActD) and cycloheximide (CHX) on lovastatin-induced cytotoxicity and apoptosis in MCF-7, SCC9 and SCC25 cell lines. A MTT enzyme activity after exposure to 0–70 μM lovastatin alone or in combination with 0.5 ng/ml ActD or 0.1 μM CHX for 48 h was analyzed. The addition of ActD or CHX appeared to inhibit lovastatin-induced cytotoxicity in these cell lines; B flow cytometric analysis of solvent control, 10 μM lovastatin and 50 μM lovastatin with the addition of 5 ng/ml ActD or 0.1 μM CHX for 48 h was evaluated. Both ActD and CHX inhibited lovastatin-induced apoptosis in SCC25 cells

Inhibition of EGFR potentiates lovastatin-induced apoptosis

A number of therapeutic approaches in HNSCC revolve around targeting and inhibiting the function of EGFR including blocking antibodies that inhibit ligand binding and specific tyrosine kinase inhibitors (Mendelsohn and Baselga 2000). Due to the essential role of mevalonate metabolites in transducing EGFR signals, in this study we evaluated the effects of the AG1478 EGFR tyrosine kinase inhibitor (Partik et al. 1999; Zhu et al. 2001) on lovastatin-induced apoptosis and the potential of lovastatin to target EGFR function. Using MTT analysis, AG1478-induced cytotoxicity was pronounced in the HNSCC cell lines SCC9 and SCC25, while the MCF-7 cells, where EGFR expression is not detected by Western blot analysis, showed toxicity only at high doses (200 μM) (Fig. 6A). At these doses, this drug can also affect other cellular targets (Partik et al. 1999; Zhu et al. 2001). Employing the 10-μM AG1478 concentration that did not affect MCF-7 MTT activity, this agent potentiated the cytotoxic effects of lovastatin in the SCC9 and SCC25 cell lines, but not the MCF-7 cell line (Fig. 6A). Although this effect was not additive, AG1478 was the only agent tested that consistently showed augmentation of lovastatin-induced cytotoxicity in our model systems.

Fig. 6. A.

Fig. 6. A

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MTT analysis of AG1478 (EGFR tyrosine kinase inhibitor) exposure alone and in combination with lovastatin for 48 h. The SCC9 and SCC25 cell lines were sensitive to the cytotoxic effects of AG1478 in comparison to MCF-7. The combination of lovastatin and 10 μM AG1478 showed an additive effect in the SCC cell lines, but not in MCF-7. B Western blot analysis of EGFR and activated EGFR, visualized by the phospho-specific antibody that detects phospho-tyrosine at 1068 site of the receptor. Following addition of EGF for 15 min, the activation of EGFR was inhibited by lovastatin in a time-dependent manner. By 24 h treatment, the autophosphorylation of EGFR was abrogated. Therefore, lovastatin treatment appears to target the function of EGFR

The co-operativity of AG1478 and lovastatin may result from the ability of both of these agents to target EGFR signaling. We analyzed the ability of lovastatin to affect the activation of EGFR. In this experiment, we treated the SCC9 and SCC25 cell lines with 10 μM lovastatin for various time points up to 24 h with or without EGF addition to stimulate EGFR. Activated EGFR was visualized by Western blot analysis using a phosphospecific antibody that recognizes a phosphotyrosine at the 1068 amino acid site of this receptor (Bishayee et al. 1999) (Fig. 6B). Upon ligand stimulation, this site is autophosphorylated and is one of the main tyrosine phosphorylated residues that facilitates the transduction of signal from this receptor (Bishayee et al. 1999). Following 12- or 24-h treatment with lovastatin, EGF stimulated autophosphorylation of EGFR was significantly inhibited in both the SCC25 and SCC9 cell lines, respectively (Fig. 6B). The MCF-7 cells did not express detectable levels of EGFR or activated EGFR by Western blot analysis under these conditions (data not shown). Taken together, these results suggest that lovastatin may target EGFR function and that this may play a role in the anti-cancer properties of lovastatin.

Based on the observation that lovastatin inhibits autophosphorylation of EGFR in a time-dependent manner, we evaluated the effects of a 24-h pretreatment of these cell lines followed by combinations with AG1478. MTT analysis demonstrated a significant additive effect of this treatment regimen in SCC9 and SCC25 with no effect in MCF-7 cells (Fig. 7A). This combination induced a potent cytotoxic response in the HNSCC cells tested with a dramatic reduction of 80% in cell viability in the SCC9 and SCC25 cell with negligible effects on MCF-7 cells (Fig. 7A). Flow cytometric analysis showed that this additive cytotoxic effect was mediated by the ability of AG1478 to potentiate the apoptotic effects of lovastatin in the SCC25 cell line (Fig. 7B).

Fig. 7.

Fig. 7.

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MTT assay and flow cytometric analysis of MCF-7, SCC9 and SCC25 cell lines pretreated with either solvent control (control and 10 μM AG1478 treatments) or 10 μM lovastatin (10 μM lovastatin and lovastatin+AG1478 treatments) followed by 48-h treatment with lovastatin alone, AG1478 alone or their combination. The combination of lovastatin and AG1478 treatments demonstrated a significant additive effect compared to either treatment alone (paired t-test) in the SCC9 and SCC25 HNSCC cell lines, but not in the MCF-7 cells. Under similar conditions, flow cytometric analyses showed a marked potentiation of lovastatin-induced apoptosis with AG1478 with minimal effects on apoptosis or cell cycle in MCF-7 cells

Discussion

Failure of cells to adequately control their proliferation, differentiation, cell survival and/or apoptosis contribute to neoplastic transformation (Fisher 1994). Apoptosis is a highly regulated cellular process that can be activated as a result of aberrant proliferation or differentiation, abrogation of cell survival signals or in response to cellular damage resulting in programmed cell death (Fisher 1994). Chemotherapeutic agents and radiation target these cellular fates, particularly proliferation, resulting in the induction of an apoptotic response (Fisher 1994). More recently, novel agents and therapeutic approaches that regulate cell survival and signaling inducing apoptosis have been developed as a direct result in the progress of elucidating the cellular components of these pathways (Penn 2001). We recently identified lovastatin, a potent inhibitor of HMG-CoA reductase, as an agent that can trigger tumor-specific apoptosis, particularly in HNSCC derived cell lines (Dimitroulakos et al. 1999; Dimitroulakos et al. 2001; Dimitroulakos and Yeger 1996). Lovastatin is also a potent inhibitor of tumor cell growth (Keyomarsi et al. 1991) and as such has been evaluated as a potential anti-cancer therapeutic agent (Thibault et al. 1996). Based on these clinical studies, it has become apparent that the most relevant application of this agent would be as part of a combined modality approach.

In this study, we evaluated the potential of standard chemotherapeutics that have demonstrated activity in HNSCC to potentiate the apoptotic effects of lovastatin in preclinical in vitro studies. The chemotherapeutics tested did not significantly augment the apoptotic effects of lovastatin in the HNSCC cell lines tested, and the higher cytotoxic levels of cisplatin and 5-FU apparently inhibited lovastatin-induced apoptosis. Furthermore, perturbing transcription or translation in these cells also inhibited lovastatin-induced apoptosis, suggesting that these cellular processes that are also affected by these chemotherapeutics (Cohen and Lippard 2001; Grem 2000) are required for the cytotoxic effects of lovastatin.

Lovastatin targets the rate-limiting enzyme of the mevalonate pathway, resulting in a depletion of its biosynthetic end products (Corsini et al. 1995; Hunninghake 1992). Mevalonate metabolites play significant roles in the post-translational modifications of a wide variety of key cellular proteins that are critical for their function. For example, dolichol is a molecular chaperone that modulates the N-linked glycosylation of a number of cell surface proteins including the EGFR (Bishayee 2000; Slieker et al. 1988). Glycosylation of EGFR is critical for its proper conformation and function as well as for efficient ligand binding and activation (Bishayee 2000; Slieker et al. 1988). As well, a wide variety of proteins are isoprenylated, the post-translational addition of a farnesyl or geranylgeranyl moiety that is required for their membrane localization and activity. Isoprenylated proteins include the members of the ras, rho and rab family of proteins (Gibbs et al. 1994; Pruitt and Der 2001; Seabra et al. 2002) that play key roles in transducing signals from a number of receptor tyrosine kinases including the EGFR (Gschwind et al. 2001; Pruitt and Der 2001; Takai et al. 2001). Therefore, inhibiting the mevalonate pathway has the potential to target EGFR and/or its signaling cascade.

The EGFR is a regulator of growth, differentiation and survival of epithelial cells and is also involved in the development and progression of cancers derived from these tissues, including HNSCC (Mendelsohn and Baselga 2000). Targeting EGFR function has been an intensive focus of anti-cancer therapeutic approaches in HNSCC (Arteaga and Johnson 2001; Mendelsohn and Baselga 2000). Due to the potential of statins to target EGFR downstream signaling and the availability of agents that target the EGFR more directly, we evaluated the potential synergy of combining these approaches. AG1478 is a potent and specific inhibitor of the EGFR tyrosine kinase, targeting the signal transduction pathway at the level of the receptor (Partik et al. 1999; Zhu et al. 2001). AG1478 demonstrated dramatic additive apoptotic effects with lovastatin in HNSCC cells. Furthermore, lovastatin displayed the potential to target EGFR function in a time-dependent manner, whereas AG1478 binds to the ATP binding site of this receptor directly inhibiting autophosphorylation (Partik et al. 1999; Zhu et al. 2001). These results indicate that these two therapeutic approaches may act co-operatively to target this receptor, albeit through different mechanisms. A number of therapeutic approaches targeting EGFR are currently in clinical trials, including ligand binding inhibitor antibodies (Mendelsohn and Baselga 2000) and various tyrosine kinase inhibitors of the receptor (Arteaga and Johnson 2001; Herbst 2002). The feasibility of combining statins with these clinically relevant EGFR inhibitors should be tested in well-established xenograft murine models of squamous cell carcinomas. These experiments were beyond the scope of this study. The combination of statins and EGFR tyrosine kinase inhibitors is an attractive therapeutic approach in HNSCC. Both agents alone demonstrate significant pre-clinical efficacy in HNSCC cells (Dimitroulakos et al. 2002; Dimitroulakos et al. 2001; Herbst 2002; Mendelsohn and Baselga 2000), and clinical trials have shown a different spectrum of toxicities (Arteaga and Johnson 2001; Herbst 2002; Thibault et al. 1996). Combining these therapeutic approaches will be invaluable in HNSCC patients. The ability of lovastatin to inhibit EGFR function is intriguing and requires further study to elucidate its mechanism and potentially allow for more refined therapeutic approaches using combinations of these agents.

Acknowledgements

Support from Cancer Care Ontario (JD) and the Ottawa Regional Cancer Centre Foundation (JD) is greatly appreciated. We wish to thank Sean Hopkins and Dr. Samy El-Sayed for helpful discussions and critically reviewing this manuscript. We wish to thank Apotex and the Ottawa Regional Cancer Centre Pharmacy for generously providing reagents used in this study.

Abbreviations

HNSCC

head and neck squamous cell carcinoma

EGFR

epidermal growth factor receptor

5-FU

5-fluorouracil

HMG-CoA

3-hydroxy-3-methylglutaryl coenzyme A

ActD

actinomycin D

CHX

cycloheximide

References

  1. Agarwal B, Bhendwal S, Halmos B, Moss SF, Ramey WG, Holt PR (1999) Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells. Clin Cancer Res 5:2223–2229 [PubMed] [Google Scholar]
  2. Arteaga CL, Johnson DH (2001) Tyrosine kinase inhibitors-ZD1839 (Iressa). Curr Opin Oncol 13:491–498 [DOI] [PubMed] [Google Scholar]
  3. Atula T, Silvoniemi P, Kurki T, Varpula M, Grenman R (1997) The evaluation and treatment of the neck in carcinoma of the oral cavity. Acta Otolaryngol [Suppl 5] 29:223–225 [DOI] [PubMed] [Google Scholar]
  4. Bishayee S (2000) Role of conformational alteration in the epidermal growth factor receptor (EGFR) function. Biochem Pharmacol 60:1217–1223 [DOI] [PubMed] [Google Scholar]
  5. Bishayee A, Beguinot L, Bishayee S (1999) Phosphorylation of tyrosine 992, 1068 and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail. Mol Biol Cell 10:525–536 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boring C, Squire TS, Tong T (1992) Cancer statistics. CA Cancer J Clin 42:19–38 [DOI] [PubMed] [Google Scholar]
  7. Boring CC, Squire TS, Tong T, Montgomery S (1994) Cancer statistics. CA Cancer J Clin 44:7–26 [DOI] [PubMed] [Google Scholar]
  8. Cohen SM, Lippard SJ (2001) Cisplatin: from DNA damage to cancer chemotherapy. Prog Nucleic Acid Res Mol Biol 67:93–130 [DOI] [PubMed] [Google Scholar]
  9. Corsini A, Maggi FM, Catapano AL (1995) Pharmacology of competitive inhibitors of HMG-CoA reductase. Pharmacol Res 31:9–27 [DOI] [PubMed] [Google Scholar]
  10. Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, Traganos F (1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13:795–808 [DOI] [PubMed] [Google Scholar]
  11. Dimitroulakos J, Yeger H (1996) HMG-CoA reductase mediates the biological effects of retinoic acid on human neuroblastoma cells: lovastatin specifically targets P-glycoprotein-expressing cells. Nat Med 2:326–333 [DOI] [PubMed] [Google Scholar]
  12. Dimitroulakos J, Nohynek D, Backway KL, Hedley DW, Yeger H, Freedman MH, Minden MD, Penn LZ (1999) Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis: a potential therapeutic approach. Blood 93:1308–1318 [PubMed] [Google Scholar]
  13. Dimitroulakos J, Ye LY, Benzaquen M, Moore MJ, Kamel-Reid S, Freedman MH, Yeger H, Penn LZ (2001) Differential sensitivity of various pediatric cancers and squamous cell carcinomas to lovastatin-induced apoptosis: therapeutic implications. Clin Cancer Res 7:158–167 [PubMed] [Google Scholar]
  14. Dimitroulakos J, Marhin WH, Tokunaga J, Irish J, Gullane P, Penn LZ, Kamel-Reid S (2002) Microarray and biochemical analysis of lovastatin-induced apoptosis of squamous cell carcinomas. Neoplasia 4:337–346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dumontet C, Sikic BI (1999) Mechanisms of, action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death. J Clin Oncol 17:1061–1070 [DOI] [PubMed] [Google Scholar]
  16. Feleszko W, Jakobisiak M (2000) Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells. Clin Cancer Res 6:1198–1199 [PubMed] [Google Scholar]
  17. Feleszko W, Mlynarczuk I, Balkowiec-Iskra EZ, Czajka A, Switaj T, Stoklosa T, Giermasz A, Jakobisiak M (2000) Lovastatin potentiates antitumor activity and attenuates cardiotoxicity of doxorubicin in three tumor models in mice. Clin Cancer Res 6:2044–2052 [PubMed] [Google Scholar]
  18. Feleszko W, Mlynarczuk I, Olszewska D, Jalili A, Grzela T, Lasek W, Hoser G, Korczak-Kowalska G, Jakobisiak M (2002) Lovastatin potentiates antitumor activity of doxorubicin in murine melanoma via an apoptosis-dependent mechanism. Int J Cancer 100:111–118 [DOI] [PubMed] [Google Scholar]
  19. Fisher DE (1994) Apoptosis in cancer therapy: crossing the threshold. Cell 78:539–542 [DOI] [PubMed] [Google Scholar]
  20. Forastiere AA, Shank D, Neuberg D, Taylor SGt, DeConti RC, Adams G (1998) Final report of a phase II evaluation of paclitaxel in patients with advanced squamous cell carcinoma of the head and neck: an Eastern Cooperative Oncology Group trial (PA390). Cancer 82:2270–2274 [PubMed] [Google Scholar]
  21. Gibbs JB, Oliff A, Kohl NE (1994) Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic. Cell 77:175–178 [DOI] [PubMed] [Google Scholar]
  22. Goldstein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425–430 [DOI] [PubMed] [Google Scholar]
  23. Grem JL (2000) 5-Fluorouracil: forty-plus and still ticking. A review of its preclinical and clinical development. Invest New Drugs 18:299–313 [DOI] [PubMed] [Google Scholar]
  24. Gschwind A, Zwick E, Prenzel N, Leserer M, Ullrich A (2001) Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene 20:1594–1600 [DOI] [PubMed] [Google Scholar]
  25. Herbst RS (2002) ZD1839: targeting the epidermal growth factor receptor in cancer therapy. Expert Opin Investig Drugs 11:837–849 [DOI] [PubMed] [Google Scholar]
  26. Hoffmann T, Hafner D, Ballo H, Haas I, Bier H (1997) Antitumor activity of anti-epidermal growth factor receptor monoclonal antibodies and cisplatin in ten human head and neck squamous cell carcinoma lines. Anticancer Res 17:4419–4425 [PubMed] [Google Scholar]
  27. Hunninghake DB (1992) HMG-CoA reductase inhibitors. Curr Opin Lipidol 3:22–28 [DOI] [PubMed] [Google Scholar]
  28. Kawata S, Yamasaki E, Nagase T, Inui Y, Ito N, Matsuda Y, Inada M, Tamura S, Noda S, Imai Y, Matsuzawa Y (2001) Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial. Br J Cancer 84:886–891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kelland LR, McKeage MJ (1994) New platinum agents. A comparison in ovarian cancer. Drugs Aging 5:85–95 [DOI] [PubMed] [Google Scholar]
  30. Keyomarsi K, Sandoval L, Band V, Pardee AB (1991) Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res 51:3602–3609 [PubMed] [Google Scholar]
  31. Kim SY, Han IS, Yu HK, Lee HR, Chung JW, Choi JH, Kim SH, Byun Y, Carey TE, Lee KS (1998) The induction of P450-mediated oxidation of all-trans retinoic acid by retinoids in head and neck squamous cell carcinoma cell lines. Metabolism 47:955–958 [DOI] [PubMed] [Google Scholar]
  32. Koutsoukos AD, Rubinstein LV, Faraggi D, Simon RM, Kalyandrug S, Weinstein JN, Kohn KW, Paull KD (1994) Discrimination techniques applied to the NCI in vitro anti-tumour drug screen: predicting biochemical mechanism of action. Stat Med 13:719–730 [DOI] [PubMed] [Google Scholar]
  33. Lamont EB, Vokes EE (2001) Chemotherapy in the management of squamous-cell carcinoma of the head and neck. Lancet Oncol 2:261–269 [DOI] [PubMed] [Google Scholar]
  34. Mendelsohn J, Baselga J (2000) The EGF receptor family as targets for cancer therapy. Oncogene 19:6550–6565 [DOI] [PubMed] [Google Scholar]
  35. Monks A, Scudiero DA, Johnson GS, Paull KD, Sausville EA (1997) The NCI anti-cancer drug screen: a smart screen to identify effectors of novel targets. Anticancer Drug Des 12:533–541 [PubMed] [Google Scholar]
  36. Partik G, Hochegger K, Schorkhuber M, Marian B (1999) Inhibition of epidermal-growth-factor-receptor-dependent signalling by tyrphostins A25 and AG1478 blocks growth and induces apoptosis in colorectal tumor cells in vitro. J Cancer Res Clin Oncol 125:379–388 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Penn LZ (2001) Apoptosis modulators as cancer therapeutics. Curr Opin Investig Drugs 2:684–692 [PubMed] [Google Scholar]
  38. Piacentini M, Fesus L, Melino G (1993) Multiple cell cycle access to the apoptotic death programme in human neuroblastoma cells. FEBS Letts 320:150–154 [DOI] [PubMed] [Google Scholar]
  39. Pinto HA, Jacobs C (1991) Chemotherapy for recurrent and metastatic head and neck cancer. Hematol Oncol Clin North Am 5:667–686 [PubMed] [Google Scholar]
  40. Pruitt K, Der CJ (2001) Ras and Rho regulation of the cell cycle and oncogenesis. Cancer Lett 171:1–10 [DOI] [PubMed] [Google Scholar]
  41. Raymond E, Chaney SG, Taamma A, Cvitkovic E (1998) Oxaliplatin: a review of preclinical and clinical studies. Ann Oncol 9:1053–1071 [DOI] [PubMed] [Google Scholar]
  42. Seabra MC, Mules EH, Hume AN (2002) Rab GTPases, intracellular traffic and disease. Trends Mol Med 8:23–30 [DOI] [PubMed] [Google Scholar]
  43. Slieker LJ, Martensen TM, Lane MD (1988) Biosynthesis of the epidermal growth factor receptor: post-translational glycosylation-independent acquisition of tyrosine kinase autophosphorylation activity. Biochem Biophys Res Commun 153:96–103 [DOI] [PubMed] [Google Scholar]
  44. Takai Y, Sasaki T, Matozaki T (2001) Small GTP-binding proteins. Physiol Rev 81:153–208. [DOI] [PubMed] [Google Scholar]
  45. Thibault A, Samid D, Tompkins AC, Figg WD, Cooper MR, Hohl RJ, Trepel J, Liang B, Patronas N, Venzon DJ, Reed E, Myers CE (1996) Phase 1 study of lovastatin, an inhibitor of the mevalonate pathway, in patients with cancer. Clinical Cancer Res 2:483–491 [PubMed] [Google Scholar]
  46. Trimmer EE, Essigmann JM (1999) Cisplatin. Essays Biochem 34:191–211 [DOI] [PubMed] [Google Scholar]
  47. van Gog FB, Brakenhoff RH, Stigter-van Walsum M, Snow GB, van Dongen GA (1998) Perspectives of combined radioimmunotherapy and anti-EGFR antibody therapy for the treatment of residual head and neck cancer. Int J Cancer 77:13–18 [DOI] [PubMed] [Google Scholar]
  48. Vokes EE, Weichselbaum RR, Lippman SM, Hong WK (1993) Head and neck cancer. N Engl J Med 328:184–194 [DOI] [PubMed] [Google Scholar]
  49. Wittes RE, Cvitkovic E, Shah J, Gerold FP, Strong EW (1977) CIS-Dichlorodiammineplatinum(II) in the treatment of epidermoid carcinoma of the head and neck. Cancer Treat Rep 61:359–366 [PubMed] [Google Scholar]
  50. Zhu XF, Liu ZC, Xie BF, Li ZM, Feng GK, Yang D, Zeng YX (2001) EGFR tyrosine kinase inhibitor AG1478 inhibits cell proliferation and arrests cell cycle in nasopharyngeal carcinoma cells. Cancer Lett 169:27–32 [DOI] [PubMed] [Google Scholar]

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