Farrerol Induces Cancer Cell Death via ERK Activation in SKOV3 Cells and Attenuates TNF-α-Mediated Lipolysis

Abstract

Farrerol (FA) is a flavanone isolated from the Chinese herbal medicine "Man-shan-hong" (Rhododendron dauricum L.). In the present study, FA decreased the viability of SKOV3 cells in a dose- and time-dependent manner, and it induced G2/M cell cycle arrest and cell apoptosis. Cell cycle distribution analysis via flow cytometry showed that FA decreased G1 populations and increased G2/M populations in SKOV3 cells. Additionally, Western blotting confirmed an increase in the expression level of proteins involved in the cell cycle, e.g., CDK and cyclins. FA-induced apoptosis in SKOV3 cells was also investigated using a TUNEL assay, and increased expression levels of proapoptotic factors, including Caspase-3 and poly ADP ribose polymerase (PARP), through the Extracellular signal-regulated kinase (ERK)/MAPK pathway were investigated. Proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-1) have been identified as a driver of the pathological mechanisms underlying involuntary weight loss and impaired physical function, i.e., cachexia, during cancer; in the present study, we showed that farrerol attenuates TNF-α-induced lipolysis and increases adipogenic differentiation in 3T3-L1 cells. Thus, farrerol could potentially be used as an anticancer agent or anticachetic drug.

Keywords: adipogenesis; antitumor effect; cachexia; cell cycle arrest; farrerol; ovarian cancer.

Figure 1

The chemical structure of farrerol.

Figure 2

Farrerol (FA) inhibits cell proliferation of SKOV3 cells through G2/M cell cycle arrest. (a) Crystal violet-stained cell images of SKOV3 cells after treatment with the indicated concentration of FA for 24 and 48 h. (b) The cell viability of SKOV3 and EA.hy926 on FA treatment cells was measured via an MTT assay. Data are means ± standard deviation from three independent experiments (*, p < 0.05). (c) SKOV3 cells were treated with FA (0, 40, 80, or 160 μM) for 24 or 48 h, harvested, and digested with RNase. Cellular DNA was stained with propidium iodide, and the cell cycle distribution was analyzed using flow cytometry. (d) Cell cycle distribution data.

Figure 3

Effect of farrerol (FA) on cell cycle regulatory proteins in SKOV3 cells. (a) SKOV3 cells were treated with FA (0, 40, 80, or 160 μM) for 24 or 48 h and then harvested. The cell lysates were prepared, subjected to SDS-PAGE, and analyzed by Western blotting. The expression of Cyclin D1, CDK4, Cyclin E1, CDK2, Cyclin B1, Cyclin A, and Cdc2 was determined; the expression of β-actin was used to verify equal loading of the samples. (b) Representative image of the Western blots. Data are means ± standard deviation from three independent experiments (*, p < 0.05).

Figure 4

Effect of farrerol (FA) on the induction of apoptosis in SKOV3 cells. (a) SKOV3 cells were treated with the indicated concentration of FA for 24 or 48 h. Apoptotic cells were detected using a Annexin V-FITC/PI staining assay. (b) Quantification of apoptotic cell ratio of Annexin V/PI staining assay. (c) SKOV3 cells were treated with FA for 24 or 48 h. The cells were lysed and total proteins were separated by SDS-PAGE. Equal loading was confirmed by the quantification of β-actin. (d) Representative images of western blots. Data are means ± SD from three independent experiments (*, p < 0.05).

Figure 5

Apoptotic effect of farrerol (FA) in SKOV3 was mediated by the ERK MAPK pathway. (a) Expression levels of MAPK in FA-treated SKOV3 cells were detected using Western blotting. (b) The viability of SKOV3 cells following treatment with each MAPK inhibitor with or without FA (160 μM) was measured via an MTT assay. (c) Caspase-3 activity of SKOV3 cells following treatment with each MAPK inhibitor with or without FA (160 μM), as measured by a colorimetric Caspase-3 activity assay. (d) Expression levels of PARP, cleaved PARP, ERK, and phospho-ERK in FA-treated SKOV3 cells with or without PD98059, as detected using Western blotting. (e) Expression levels of the cleaved PARP/PARP ratio. (f) Expression levels of the p-ERK/ERK ratio. All data are means ± standard deviation from three independent experiments.

Figure 6

Farrerol (FA) reverses TNF-α induced lipid wasting in differentiated 3T3-L1 cells. (a) Differentiated 3T3-L1 cells were treated with TNF-α (20 ng/mL) with or without FA (15 or 30 µM) for 48 h. The representative images of the Oil Red O (ORO)-stained-cells in each indicated group are shown. (b) The ORO staining in the cells was dissolved in Propan-2-ol and measured. (c) Expression levels of PGC1a, UCP-1, and PPARγ in differentiated 3T3-L1 cells treated with TNF-α with or without FA for 48 h were detected using Western blotting. (d) The expression levels of PGC1a, UCP-1, and PPARγ are shown. Equal loading was confirmed by the quantification of β-actin. Data represent the means ± standard deviation from three independent experiments.

Figure 7

Effects of farrerol (FA) on lipid accumulation, triglyceride content, and adipogenic differentiation in 3T3-L1 cells. (a) First, 3T3-L1 preadipocytes were treated with differentiation medium in the presence or absence of FA at 15 or 30 μg/mL for 8 days. Lipid accumulation was quantified by staining with Oil Red O (ORO) solution and absorbance was measured at 495 nm. Differentiated 3T3-L1 cells photographed at 100× magnification after ORO staining. (b) Quantification of ORO-stained intracellular lipid content. (c) Intracellular triglyceride content quantified by a triglyceride assay kit and assessed at 570 nm. Preadipocytes were used as negative controls (NC), whereas fully differentiated adipocytes were used as positive controls (PC). (d) Cell viabilities were determined with an MTT assay. (e) The 3T3-L1 preadipocytes were treated with FA at the indicated concentrations for 24 or 48 h. The relative mRNA expression levels of PPARγ, C/EBPα, adiponectin, HSL, and LPL were assessed by real-time PCR. Each experiment was repeated in triplicate. Bars represent means ± standard error (*, p < 0.01 compared to control group).