Isorhamnetin inhibits cell proliferation and induces apoptosis in breast cancer via Akt and mitogen‑activated protein kinase kinase signaling pathways

Abstract

Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-κB inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways.

Figure 1

Isorhamnetin inhibits proliferation and induces apoptosis of breast cancer cells. (A) The cells were treated with various concentrations of isorhamnetin for 72 h, cell proliferation was determined using a Cell Counting kit-8 (CCK-8) assay and the IC₅₀ was calculated. ^*P<0.05, IC₅₀ of isorhamnetin in various cancer cells as compared with the MCF10A cells. (B) Isorhamnetin induced apoptosis of the MCF7 and MDA-MB-468 cells following a 48-h treatment. The upper right quandrant shows early apoptotic cells, the lower right the late apoptotic cells, the upper left quadrant indicates cell debris and the lower left the viable cells. IC₅₀, half maximal inhibitory concentration; PI, propidium iodide.

Figure 2

Effects of isorhamnetin on the cell signaling cascade. (A) Isorhamnetin inhibited the phosphorylation of Akt, mTOR, MEK1/2 and ERK1/2, but not of EGFR (12-h treatment). The protein expression levels were quantified by Image J, and presented as the relative expression levels to the control. (B) Isorhamnetin increased the expression level of Bax and cleaved caspase 3, and decreased the expression level of Bcl-2 and Bcl-xL (12-h treatment); (C) Isorhamnetin decreased the nuclear translocation of NF-κB (24-h treatment). p, phosphorylated; mTOR, mammalian target of rapamycin; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; EGFR, epidermal growth factor receptor; PI3K, phosphoinositide 3-kinase; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-associated protein X; Bcl-2-xL, Bcl-extra large; NF-κB, nuclear factor-κB. ^*P<0.05, vs. the control group.

Figure 3

Induction of apoptosis was attenuated by the Akt and MEK inhibitors in the MCF7 cells. (A) Induction of caspase-3 cleavage was attenuated by the Akt inhibitor, perifosine (12-h treatment). The protein expression levels were quantified by Image J, and presented as the relative expression levels to the control. (B) Induction of caspase-3 cleavage was attenuated by the MEK inhibitor, PD184352 (12-h treatment). ^*P<0.05, vs. the control group. (C) Induction of cell apoptosis was attenuated by the Akt inhibitor, perifosine (48-h treatment). (D) Induction of cell apoptosis was attenuated by the MEK inhibitor, PD184352 (48-h treatment). MEK, mitogen-activated protein kinase kinase; Con, control; PI, propidium iodide; PD, PD184352; Per, perifosine; Iso, isorhamnetin. The upper right quadrant shows the early apoptotic cells, the lower right the late apoptotic cells, the upper left quadrant indicates cell debris and the lower left the viable cells.

Figure 4

Apoptotic levels were decreased following treatment with EGF in the MCF7 cells. EGF induced the phosphorylation of EGFR, Akt and MEK, which reversed the inhibitory effects of isorhamnetin on Akt and MEK phosphorylation, and attenuated the levels of cell apoptosis induced by isorhamnetin, as shown by decreased levels of cleaved caspase-3. MEK, mitogen-activated protein kinase kinase; EGFR, epidermal growth factor receptor.

Figure 5

JSH-23, the NF-κB inhibitor, did not contribute to the induction of apoptosis by isorhamnetin in the MCF7 cells. (A) Induction of caspase-3 cleavage was not increased by treatment with JSH-23 (12-h treatment); (B) Induction of cell apoptosis was not augmented by the NF-kB inhibitor, JSH-23 (48 h treatment). NF-κB, nuclear factor-κB; Con, control; PI, propidium iodide; JSH, JSH-23; Iso, isorhamnetin.