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

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 microM 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 microM 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.

Fig. 1.

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

Fig. 2.

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)

Fig. 3.

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

Fig. 4.

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

Fig. 5.

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

Fig. 6. A

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

Fig. 7.

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