Genistein synergizes with arsenic trioxide to suppress human hepatocellular carcinoma

Abstract

Arsenic trioxide (ATO) is of limited therapeutic benefit for the treatment of solid tumors. Genistein exhibits anticancer and pro-oxidant activities, making it a potential candidate to enhance the efficacy of ATO whose cytotoxicity is oxidation-sensitive. This study sought to determine whether genistein synergizes with ATO to combat hepatocellular carcinoma (HCC). Three human HCC cell lines, namely HepG2, Hep3B, and SK-Hep-1, were incubated with ATO, genistein, or ATO + genistein. The cells were also pretreated with antioxidant agents N-acetyl-L-cysteine (NAC) or butylated hydroxyanisole (BHA). Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (DeltaPsim), expression of Bcl-2, Bax, caspase-9, and -3, and release of cytochrome c into the cytosol were examined. The synergistic effect of ATO and genistein was also assessed using HepG2 xenografts subcutaneously established in BALB/c nude mice. The results show that genistein synergized with ATO to reduce viability, induce apoptosis, and diminish the DeltaPsim of cells. The combination therapy down-regulated Bcl-2 expression, up-regulated Bax expression, enhanced the activation of caspase-9 and -3, and increased the release of cytochrome c. The synergistic effect of ATO and genistein was diminished by pretreatment with NAC or BHA. Genistein increased the production of intracellular ROS, while ATO had little effect. Genistein synergized with a low dose of ATO (2.5 mg/kg) to significantly inhibit the growth of HepG2 tumors, and suppress cell proliferation and induce apoptosis in situ. There were no obvious side effects, as seen with a high dose of ATO (5 mg/kg). Combining genistein with ATO warrants investigation as a therapeutic strategy to combat HCC.

Figure 1

Cell growth in vitro. As indicated, HepG2, Hep3B, and SK‐Hep‐1 cells were incubated with arsenic trioxide (ATO), genistein (GEN), ATO + GEN, or pretreated with N‐acetyl‐L‐cysteine (NAC) or butylated hydroxyanisole (BHA) followed by ATO + GEN, for 72 h. Untreated cells served as the control. Cell viability was determined using a Cell Counting Kit‐8 (CCK‐8) assay to calculate the growth index. *Significant reduction in the growth index from control; **highly significant difference at P < 0.001 from control; †significant reduction from same dose ATO treatment; ‡significant increase from 2 μm ATO + GEN treatment.

Figure 2

Cell apoptosis in vitro. (A) HepG2, Hep3B, and SK‐Hep‐1 cells were treated with arsenic trioxide (ATO), genistein (GEN) ATO + GEN, or pretreated with N‐acetyl‐L‐cysteine (NAC) or butylated hydroxyanisole (BHA) followed by ATO + GEN, for 48 h. Untreated cells served as the control. The cells were stained with Annexin V/PI, and subjected to flow cytometry to measure the apoptosis rate (%). *Significant increase in apoptosis rate from control; **highly significant difference at P < 0.001 from control; †significant increase from same dose ATO treatment; ‡significant reduction from 2 μm ATO + GEN treatment. (B) Representative histograms are shown for cytometrically analyzed cells. (C) Representative photographs of control and ATO + GEN‐treated cells stained with Annexin V/PI and viewed by laser scanning confocal microscopy (scale bar, 20 μm).

Figure 3

Levels of intracellular reactive oxygen species (ROS) in vitro. (A) HepG2, Hep3B, and SK‐Hep‐1cells were treated with 2 μm arsenic trioxide (ATO), genistein (GEN) (15 μm for HepG2 and SK‐Hep‐1, 20 μm for Hep3B), ATO + GEN, or pretreated with N‐acetyl‐L‐cysteine (NAC) or butylated hydroxyanisole (BHA) followed by GEN or ATO + GEN, for 12 h. Untreated cells served as the control. The cells were incubated with 2′,7′‐dichlorodihydrofluorescein‐diacetate (DCFHDA), then subjected to flow cytometry to measure levels of intracellular ROS, represented by dichlorofluorescein (DCF) fluorescence. *Significant increase in DCF fluorescence from control; **highly significant difference from control at P < 0.001; †significant increase from ATO treatment; #significant reduction from GEN treatment; ‡significant reduction from ATO + GEN treatment. (B) Representative histograms are shown for cytometrically analyzed cells stained with DCFHDA.

Figure 4

Changes in mitochondrial membrane potential in vitro. (A) HepG2, Hep3B, and SK‐Hep‐1 cells were treated with 2 μm arsenic trioxide (ATO), genistein (GEN) (15 μm for HepG2 and SK‐Hep‐1, 20 μm for Hep3B), ATO + GEN, or pretreated with N‐acetyl‐L‐cysteine (NAC) followed by ATO + GEN, for 48 h. Untreated cells served as the control. The cells were incubated with 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazole carbocyanine iodide (JC‐1), then subjected to flow cytometry to measure green and red fluorescence intensities, and the ratio of red/green fluorescence was recorded to calculate the relative ΔΨm. *Significant decrease in ΔΨm from control; **highly significant decrease from control at P < 0.001; †significant decrease from ATO treatment; ‡significant increase from ATO + GEN treatment. (B) Representative histograms are shown for cytometrically analyzed cells labeled with the JC‐1 dye.

Figure 5

Expression of apoptosis‐related proteins in vitro. (A) HepG2, Hep3B, and SK‐Hep‐1 cells were treated with 2 μm arsenic trioxide (ATO) (lane 2), genistein (GEN) (15 μm for HepG2 and SK‐Hep‐1, 20 μm for Hep3B) (lane 3), or ATO + GEN (lane 4), for 48 h. Untreated cells served as the control (lane 1). The cells were homogenized and subjected to Western blot analysis to detect the expression of Bax, Bcl‐2, pro‐caspase‐9, cleaved caspase‐9, and pro‐caspase‐3. β‐Actin served as an internal control. (B) Cytoplasmic proteins from each of the treated cells as in (A) (lanes 1 to 4) were prepared using a Mitochondria/cytosol Fractionation Kit. As an additional control, cytoplasmic proteins were prepared from cells pretreated with N‐acetyl‐l‐cysteine (NAC) followed by ATO + GEN (lane 5). The expression of cytochrome c (Cyto‐c) in the mitochondria‐depleted cytosolic fractions was examined by Western blot analysis. β‐Actin served as an internal control.

Figure 6

Genistein (GEN) synergizes with arsenic trioxide (ATO) to suppress HepG2 tumors in mice. When tumors reached ∼100 mm³ in volume, the mice received daily injections of 100 μL PBS (Control), or an equal volume of genistein at a dose of 50 mg/kg (GEN), ATO at doses of 2.5 (2.5ATO) or 5 (5ATO) mg/kg, or the combination of genistein + 2.5 mg/kg ATO (2.5ATO + GEN), for 15 days as indicated. (A) The sizes of tumors were recorded. *Significant difference in tumor volumes from control; **highly significant difference from control at P < 0.001; †significant difference from 2.5 mg/kg ATO; ‡significant difference from genistein. Blood samples were collected when the mice were euthanized on day 18, and tumors were removed. (B) The carcasses without tumors were weighed, and compared to the bodyweights on day 0 to calculate bodyweight change (%), and white blood cell (WBC) numbers were counted. (C) The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and (D) urea nitrogen (BUN) and creatinine (Cr) were measured. *Significant difference from control.

Figure 7

Genistein (GEN) synergizes with arsenic trioxide (ATO) to inhibit cell proliferation in situ. Illustrated are representative tumor sections prepared from mice that received daily injections of PBS (control) (A), 2.5 mg/kg ATO (B), genistein (C), genistein + 2.5 mg/kg ATO (D) or 5 mg/kg ATO (E), as in Figure 6. The sections were stained with an anti‐Ki‐67 Ab to detect proliferating cells. (F) Cells expressing Ki‐67 were counted to calculate the proliferation index. n, number of tumors assessed. *Significant difference in the proliferation index from control; **highly significant difference at P < 0.001 from control; †significant difference from ATO monotherapy.

Figure 8

Genistein (GEN) synergizes with arsenic trioxide (ATO) to induce cell apoptosis in situ. Illustrated are representative tumor sections prepared from mice that received daily injections of PBS (control) (A), 2.5 mg/kg ATO (B), genistein (C), genistein + 2.5 mg/kg ATO (D), or 5 mg/kg ATO (E) as in Fig. 6. The sections were stained with the TUNEL agent to visualize apoptotic cells. (F) TUNEL‐positive cells were counted to calculate the apoptosis index. n, number of tumors assessed. *Significant difference in the apoptosis index from control; **highly significant difference at P < 0.001 from control; †significant difference from ATO monotherapy. (G) The tumor tissues were homogenized and subjected to Western blot analysis to detect expression of Bax, Bcl‐2, pro‐caspase‐9, cleaved caspase‐9, and pro‐caspase‐3 (left panel). The numbering of lanes is as in Figure 5(b). β‐Actin served as an internal control. The band density was measured and compared to that of β‐actin to calculate relative band density (right panel).