Resveratrol and arsenic trioxide act synergistically to kill tumor cells in vitro and in vivo

Abstract

Background and aims: Arsenic trioxide (As2O3), which used as an effective agent in the treatment of leukaemia and other solid tumors, is largely limited by its toxicity. QT prolongation, torsades de pointes and sudden heart death have been implicated in the cardiotoxicity of As2O3. The present study was designed to explore whether the combination of As2O3 and resveratrol could generate a more powerful anti-cancer effect both in vitro and in vivo.

Materials and methods: MTT assay was performed to assess the proliferation of Hela, MCF-7 and NB4 cells. Isobolographic analysis was used to evaluate combination index values from cell viability data. The apoptosis and the cellular reactive oxygen species (ROS) level were assessed by fluorescent microscopy and flow cytometry separately in vitro. The effect of As2O3, alone and in combination with resveratrol on Hela tumor growth in an orthotopic nude mouse model was also investigated. The tumor volume and the immunohistochemical analysis of CD31, CD34 and VEGF were determined.

Results: Resveratrol dramatically enhanced the anti-cancer effect induced by As2O3 in vitro. In addition, isobolographic analysis further demonstrated that As2O3 and resveratrol generated a synergistic action. More apoptosis and ROS generation were observed in the combination treatment group. Similar synergistic effects were found in nude mice in vivo. The combination of As2O3 and resveratrol dramatically suppressed both tumor growth and angiogenesis in nude mice.

Conclusions: Combining As2O3 with resveratrol would be a novel strategy to treat cancer in clinical practice.

Figure 1. Effects of resveratrol on the cell proliferation in As₂O₃-treated Hela cells.

Hela cells were incubated with different concentrations of resveratrol (A), As₂O₃ (B) and the combination of resveratrol and As₂O₃ (C) for 48 h, the cell proliferation was then evaluated by MTT assay. Values represent Mean±SD of three independent experiments. Isobologram illustration in Hela cells for the combination of resveratrol and As₂O₃, the point (58.72, 3.45) is the IC50 of combined resveratrol and As₂O₃ (D).

Figure 2. Effects of resveratrol on the cell proliferation in As₂O₃-treated MCF-7 cells.

MCF-7 cells were incubated with different concentrations of resveratrol (A), As₂O₃ (B) and the combination of resveratrol and As₂O₃ (C) for 48 h, the cell proliferation was then evaluated by MTT assay. Values represent Mean±SD of three independent experiments. Isobologram illustration in MCF-7 cells for the combination of resveratrol and As₂O₃, the point (41.73, 3.48) is the IC50 of combined resveratrol and As₂O₃ (D).

Figure 3. Effects of resveratrol on the cell proliferation in As₂O₃-treated NB4 cells.

NB4 cells were incubated with different concentrations of the combination of resveratrol and As₂O₃ for 48 h, the cell proliferation was then evaluated by MTT assay (A). Values represent Mean±SD of three independent experiments. Isobologram illustration in NB4 cells for the combination of resveratrol and As₂O₃, the point (1.28, 15.33) is the IC50 of combined resveratrol and As₂O₃ (B).

Figure 4. Apoptosis in Hela and MCF-7 cells were evaluated by fluorescent microscopy measurements.

Hela and MCF-7 cells were treated with culture medium, resveratrol, As₂O₃, and combination of resveratrol and As₂O₃ for 48 h, then stained with AO and examined with a fluorescent microscope (×200).

Figure 5. Apoptosis in NB4 cells were quantitated by flow cytometry.

NB4 cells were treated with culture medium (A); 50 µM resveratrol (B); 3 µM As₂O₃ (C) and combination of resveratrol and As₂O₃ (D) for 48 h, then PI-FITC was carried out to investigate the apoptosis rate.

Figure 6. Change of the intracellular ROS production in Hela cells.

Hela cells were treated with culture medium (A); 50 µM resveratrol (B); 3 µM As₂O₃ (C) and resveratrol combined with As₂O₃ (D) for 48 h, then the intracellular ROS assay was performed, and captured under a fluorescence microscope (×200).

Figure 7. The tumor weight and volume in nude mice.

After 2 weeks treatment with vector (A), 16.5 mg/kg/d resveratrol (B), 5 mg/kg/d As₂O₃ (C), or combination of resveratrol and As₂O₃ (D), the tumors were excised and weighted.

Figure 8. The relative tumor volume change (compared with the tumor volume before drugs treatment) of nude mice.

35 nude mice were randomly divided into 4 groups and treated with vector, 16.5/kg/d resveratrol, 5 mg/kg/d As₂O₃, or combination of resveratrol and As₂O₃ for 2 weeks respectively. The tumor volume was monitored every day, and the tumor volume was calculated from the formula V = 1/2 (A×B²), where A was the longest diameter and B was perpendicular diameter measured by calipers.

Figure 9. HE staining of the tissue slices from xenograft tumors.

The excised tumors of mice which treated with vector (A), 16.5 mg/kg/d resveratrol (B), 5 mg/kg/d As₂O₃ (C), or combination of resveratrol and As₂O₃ (D), were fixed with formalin and sliced, then HE staining were performed.

Figure 10. The tumor vascularity was detected by the immunofluorescent staining of CD31, CD34 and VEGF.

The xenograft tumors treated with vector, 16.5/kg/d resveratrol, 5 mg/kg/d As₂O₃, or combination of resveratrol and As₂O₃ were stained for CD31, CD34 and VEGF, and then captured under a fluorescence microscope.