Quercetin-3-methyl ether inhibits lapatinib-sensitive and -resistant breast cancer cell growth by inducing G(2)/M arrest and apoptosis

Abstract

Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Here, we found that quercetin-3-methyl ether caused a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or ERKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G(2)/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G(1) phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP) in both cell lines. Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients.

Figure 1

Quercetin-3-methyl ether strongly suppresses anchorage-dependent or independent growth of SK-Br-3 and SK-Br-3-Lap R cells. (A) Quercetin-3-methyl ether inhibits anchorage-dependent cell growth in lapatinib-sensitive and -resistant breast cancer cells. Cells were treated with quercetin-3-methyl ether (0, 5, or 10 µM) or lapatinib (0.1 µM) in 10% FBS/McCoy for various times. At the end of each treatment time, cell growth was measured by MTS assay. Data are shown as means ± S.E. The asterisks (*) indicate a significant difference (p < 0.05) between groups treated with quercetin-3-methyl ether and the group treated with DMSO. (B) Quercetin-3-methyl inhibits anchorage-independent cell growth in lapatinib-sensitive and -resistant cells. Cells were treated as described under “Materials and methods” and colonies were counted under a microscope with the aid of Image-Pro Plus software (v.4). Data are shown as means ± S.E. The asterisks (**) indicate a significant difference (p < 0.001) between groups treated with quercetin-3-methyl ether or lapatinib and the group treated with DMSO.

Figure 2

Quercetin-3-methyl ether has no significant effect on Akt or MAPKs signaling in lapatinib-resistant SK-Br-3 cells. Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0, 5 or 10 µM) or lapatinib (0.1 µM) in 10% FBS/McCoy for 16 h. The levels of phosphorylated and total Akt, ERKs, JNKs and p38 proteins were determined by Western blot analysis. Semi-quantitative analysis was performed using the Image J software program.

Figure 3

Quercetin-3-methyl ether induces significant G2/M arrest in SK-Br-3 and SK-Br-3-Lap R cells, whereas lapatinib induces G1 arrest only in sensitive SK-BR-3 cells. Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0, 5, or 10 µM) or lapatinib (0.1 µM) for 16 or 48 h. Cell cycle analysis was performed using flow cytometry. Data are shown as mean percentages.

Figure 4

Quercetin-3-methyl ether activates the Chk1/p-Cdc25c (Ser216)/cyclin B1 signaling pathway in SK-Br-3 and SK-Br-3-Lap R cells. (A) Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0, 5, or 10 µM) or lapatinib (0.1 µM) in 10% FBS/McCoy for 16 h. The levels of phosphorylated and total cyclin B1, Cdk1, Cdc25c and Chk1 proteins were determined by Western blot analysis. Semi-quantitative analysis was performed using the Image J software program. (B) Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (10 µM) in 10% FBS/McCoy for 0–24 h. The level of phosphorylated Cdc25c (Ser216) was determined by Western blot analysis.

Figure 5

Quercetin-3-methyl ether induces apoptosis accompanied with an increase in cleaved caspase 3, caspase 7 and PARP in both lapatinib-sensitive and -resistant breast cancer cell lines. (A) Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0, 5, or 10 µM) or lapatinib (0.1 µM) for 48 h. Apoptosis was analyzed by flow cytometry. Data are shown as means ± S.E. The asterisk (*) indicates a significant difference (p < 0.05) between groups treated with quercetin-3-methyl ether or lapatinib and the group treated with DMSO. (B) Cells were starved in serum-free medium for 24 h and then treated with quercetin-3-methyl ether (0, 5, or 10 µM) or lapatinib (0.1 µM), in 10% FBS/McCoy for 48 h. The levels of cleaved caspase 3, caspase 7 and PARP were determined by Western blot analysis. Semi-quantitative analysis was performed using the Image J software program.