Inhibition of the MAPK pathway alone is insufficient to account for all of the cytotoxic effects of naringenin in MCF-7 breast cancer cells

Abstract

Estrogen receptor (ER) antagonists such as tamoxifen (Tam) have been used successfully to treat ER+ breast cancers for more than 30 years. Unfortunately, long term use of Tam can result in resistance. Tam resistance is associated with the activation of growth factor signaling pathways that promote cell proliferation and survival. The mitogen-activated protein kinase (MAPK), is up-regulated in Tam resistant (Tam-R) cells. Previous studies have reported that the flavanone, naringenin (Nar) can inhibit cell proliferation and induce apoptosis in ER+ breast cancer cells. Furthermore, Nar has been shown to inhibit the MAPK signaling pathways in MCF-7 cells. In this report we investigated whether inhibition of MAPK alone is mediating the effects of Nar on cell proliferation and viability. These studies will determine the mechanism of action of Nar. Tam-R MCF-7 breast cancer cells were treated with Nar or U0126, a MAPK kinase inhibitor. Our studies show that while both U0126 and Nar impaired cell proliferation and viability the combination of U0126 and Nar resulted in greater inhibition of cell viability than either compound alone. It has been previously reported that Nar can bind the ER. Our lab has also shown that Nar localizes ERα to a peri-nuclear region of the cell. Confocal microscopy revealed that in U0126 treated cells ERα displayed an even distribution across the cytoplasm as seen in untreated Tam-R cells. These studies suggest that MAPK is not the only target of Nar.

Keywords: MAPK signaling; Naringenin; Tamoxifen resistance.

Fig. 1

Nar does not impair ERα levels. Tam-R MCF-7 cells were grown in charcoal-stripped medium with 4-OHT (100 nM) in the presence or absence of Nar (200 μM) for 24, 48, and 96 h. (A) Protein lysates were subjected to SDS-PAGE and immunoblotted using antibodies against ERα and actin. (B) ERα to actin was quantified using densitometric analysis by Quantity One Software and expressed as a percent of the control. The results are representative of 3 separate experiments. Results were the means ± SEM of three independent experiments. *p < 0.05.

Fig. 2

Nar is a weak inhibitor of ERK1/2 phosphorylation. Tam-R MCF-7 cells were grown in charcoal-stripped medium with 4-OHT (100 nM) in the presence of Nar (200 μM), U0126 (10 μM) or a combination of the two for 24, 48, and 96 h. (A) Protein lysates were subjected to SDS-PAGE and immunoblotted using antibodies against phospho-ERK1/2, ERK1/2 and actin. (B) P-ERK to actin and (C) ERK to actin were quantified using densitometric analysis by Quantity One software and are expressed as a percent of the control. The results are representative of 3 separate experiments. *p < 0.05.

Fig. 3

Inhibition of ERK alone cannot explain Nar decreased cell viability. Tam-R MCF-7 cells were grown in charcoal-stripped medium with 4-OHT (100 nM) in the presence of Nar (200 μM), U0126 (10 μM) or a combination of the two for 24, 48, or 96 h. (A) Cell density (cells/mL) was determined by flow cytometry. Results are the means ± SEM of three separate experiments. Data were normalized to control. (B) Cell viability was determined by flow cytometry. Results are the means ± SEM of three independent experiments. Data were normalized to control. *p < 0.05.

Fig. 4

Nar and U0126 induce apoptosis. Tam-R MCF-7 cells were grown in charcoal-stripped medium with 4-OHT (100 nM) in the presence of Nar (200 μM), U0126 (10 μM) or a combination of the two for 24, 48, or 96 h. (A) Apoptotic cells were determined by flow cytometry. Results are the means ± SEM of three independent experiments. Data were normalized to control. (B) Protein lysates were subjected to SDS-PAGE and immunoblotted using antibodies against Caspase 7, PARP, and actin. (C) Caspase 7 and (D) PARP were quantified using densitometric analysis using Quantity One software and are expressed as a percent of the control. Results are the means ± SEM of three separate experiments. Results are normalized to control. *p < 0.05.

Fig. 5

ERK1/2 does not regulate ERα localization. Tam-R MCF-7 cells were grown in charcoal-stripped medium with 4-OHT (100 nM) in the presence of Nar (200 μM), U0126 (10 μM) or a combination of the two for 24, 48, and 96 h. (A) Cells (96 h time point) were fixed and stained with ERα antibody and DAPI and then subjected to confocal microscopy. (B) ERα localization data was quantified using intensity parameters as described in Method and Materials. Results are the means ± SEM of three separate experiments. Results are normalized to control. *p < 0.05.