Apigenin inhibits renal cell carcinoma cell proliferation

Abstract

Apigenin, a natural flavonoid found in vegetables and fruits, has antitumor activity in several cancer types. The present study evaluated the effects and mechanism of action of apigenin in renal cell carcinoma (RCC) cells. We found that apigenin suppressed ACHN, 786-0, and Caki-1 RCC cell proliferation in a dose- and time-dependent manner. A comet assay suggested that apigenin caused DNA damage in ACHN cells, especially at higher doses, and induced G2/M phase cell cycle arrest through ATM signal modulation. Small interfering RNA (siRNA)-mediated p53 knockdown showed that apigenin-induced apoptosis was likely p53 dependent. Apigenin anti-proliferative effects were confirmed in an ACHN cell xenograft mouse model. Apigenin treatment reduced tumor growth and volume in vivo, and immunohistochemical staining revealed lower Ki-67 indices in tumors derived from apigenin-treated mice. These findings suggest that apigenin exposure induces DNA damage, G2/M phase cell cycle arrest, p53 accumulation and apoptosis, which collectively suppress ACHN RCC cell proliferation in vitro and in vivo. Given its antitumor effects and low in vivo toxicity, apigenin is a highly promising agent for treatment of RCC.

Keywords: ATM signaling; DNA damage; apigenin; apoptosis; renal cell carcinoma.

Figure 1. Apigenin inhibits ACHN, 786-0, and Caki-1 cell proliferation

CCK-8 analysis showed apigenin inhibited proliferation in ACHN, 786-0, and Caki-1 RCC cells, and the normal renal proximal tubule epithelial cell line, HK-2, in a dose- and time-dependent manner (A) This analysis was repeated three times, with six replicates per experiment. Representative colony formation assay images and quantitation (B). Cell counts > 50 were defined as a colony. Representative EdU proliferation assay images using a 10× objective (C) ACHN cell proliferation rates were calculated as the ratios of EdU-staining cells to Hoechst33342-staining cells from six fields of three separate experiments. Scale bar = 25 μm. *P < 0.05, **P < 0.01 versus untreated control.

Figure 2. Apigenin treatment induces DNA damage in ACHN cells

Comet assay using 4S Green Plus dye following cell treatment with various apigenin concentrations (A) Red arrows specify cells with a “comet tail,” which indicates DNA damage. Scale bar = 25 μm. Comet formation incidences in each group were measured using 100 randomly selected cells, and DNA damage level was calculated using CaspLab software in accordance with % tail DNA (B–C). Western blotting analysis of γH2AX D. *P < 0.05, **P < 0.01 versus untreated control.

Figure 3. Apigenin causes G2/M phase cell cycle arrest and modulates cell cycle factors through ATM signaling

Apigenin induced G2/M phase cell cycle arrest in a dose-dependent manner (A–B). Western blotting analysis of ATM pathway proteins (C). *P < 0.05, **P < 0.01 versus untreated control.

Figure 4. Apigenin induces ACHN cell apoptosis, and p53 knockdown partially rescues this effect

Representative flow cytometry apoptosis assay images and quantitation of one experiment done in triplicate (A) p53 knockdown partially rescued apigenin-induced apoptosis (B) *P < 0.05, **P < 0.01.

Figure 5. Apigenin inhibits tumorigenicity in ACHN cell xenograft nude mice

Tumor xenograft model (A) ACHN cells (1 × 10⁶ in 100 μl PBS) were injected subcutaneously into the right flank of each mouse. After palpable tumors arose, apigenin (or vehicle alone) was administered at 30 mg/kg every 3 d intraperitoneally. Tumors grew more slowly in apigenin-treated mice. Harvested tumors represented smaller ones in apigenin-treated group (B). Apigenin treatment did not affect end point RBC and WBC counts (C). Apigenin treated tumors had reduced Ki-67 expression (D). Scale bar = 100 μm. Error bars represent the S.D. from five nude mice. *P < 0.05, **P < 0.01.