Synergistic inhibition of leukemia WEHI-3 cell growth by arsenic trioxide and Hedyotis diffusa Willd extract in vitro and in vivo

Abstract

Arsenic trioxide (ATO) is clinically used to treat acute promyelocytic leukemia (APL); however, the therapeutic dose of ATO may prompt critical cardiac side effects. Combination therapy may be used to improve the therapeutic efficiency. To evaluate this possibility, the present study determined the combined effects of Hedyotis diffusa Willd (HDW) extract and ATO in leukemic WEHI-3 cells. The results demonstrated that co-treatment of HDW with ATO resulted in a synergistic augmentation of cytotoxicity in cells at the concentration tested. In order to investigate the potential therapeutic application for leukemia, the combined effects of HDW and ATO were analyzed on the WEHI-3 cell-induced orthotopic leukemia animal model in vivo. The WEHI-3 cells in mice with leukemia were established by injecting murine WEHI-3 cells into BALB/c mice, and treating them with HDW and/or combined with ATO. The results indicated that HDW alone or HDW combined with ATO promoted the total survival rate of mice with leukemia, and these effects are dose-dependent. HDW alone or HDW combined with ATO did not affect the body weight, decreased the spleen weight and did not affect the liver weight. Furthermore, the results demonstrated that HDW alone or HDW combined with ATO resulted in a synergistic augmentation of apoptosis in WEHI-3 cells at the concentration tested. In order to further reveal the detailed mechanism of this synergistic effect on apoptosis, apoptosis-related proteins were also evaluated. The data revealed that HDW alone or HDW combined with ATO induced the expression of death receptor 4 (DR4) and DR5 and the activation of poly adenosine diphosphate ribose polymerase, caspase-3, -8 and -9. Furthermore, HDW alone or HDW combined with ATO decreased the expression levels of B-cell lymphoma 2, B-cell lymphoma-extra large and survivin, and increased the expression levels of Bak and t-Bid. Altogether, the results indicate that the combination of HDW with ATO may be a promising strategy used to increase the clinical efficacy of ATO in the treatment of APL.

Keywords: Hedyotis diffusa Willd; acute promyelocytic leukemia; apoptosis; arsenic trioxide; death receptor.

Figure 1.

Effect of the combination of HDW and ATO on anti-proliferation activity. WEHI-3 cells were treated with the indicated concentration of ATO or HDW, or ATO and HDW for (A) 24 and (B) 48 h, and viability was measured by the Prestoblue Cell Viability reagent assay. This experiment was repeated three times. The data represented are the mean ± standard deviation. HDW, Hedyotis diffusa Willd; ATO, arsenic trioxide.

Figure 2.

Effect of the combination of HDW and ATO on BALB/c mice following i.p. injection with WEHI-3 cells. (A) The procedure of leukemic mice establishment and treatment. (B) Mice were i.p. injected with WEHI-3 cells and subsequently orally treated with HDW, ATO or a combination of ATO and HDW for 2 weeks. All individual mice were weighed and the average weight of all mice from each group is shown. (C) The survival rate of tumor-bearing mice. The data represent the mean ± standard deviation. HDW, Hedyotis diffusa Willd; ATO, arsenic trioxide; i.p, intraperitoneal; P.O., oral gavage; DDW, double-distilled water.

Figure 3.

Effect of HDW and/or ATO on spleen or liver tissue weight in leukemic BALB/c mice. (A) Representative spleen tissue in leukemic BALB/c mice following exposure to HDW and/or ATO. BALB/c mice were intraperitoneally administered WEHI-3 leukemia cells and after 2 weeks were orally treated with HDW (100, 250 and 500 mg/kg/day) and/or ATO (5 mg/kg/day) for 2 weeks. Mice were then sacrificed, images were captured and representative spleen tissues are shown. (B) At 4 weeks, the spleen tissue weight was estimated. (Values are the mean ± standard deviation; n=6). **P<0.01 vs. normal control group; ^#P<0.05 and ^##P<0.01 vs. leukemia control group. (C) At 4 weeks, the liver tissue weight was estimated. (Values are the mean ± SD; n=6). HDW, Hedyotis diffusa Willd; ATO, arsenic trioxide; S.D. standard deviation.

Figure 4.

Effect of HDW and/or ATO on the activation of the DR4 and DR5 signaling pathway. (A) WEHI-3 cells treated with HDW at 0.4, 0.8 and 1.6 mg/ml for 48 h. (B) WEHI-3 cells were treated with 1.6 mg/ml HDW and 1.6 µM ATO or a combination of HDW and ATO for 48 h. The protein expression of DR4, DR5 and β-actin was measured by western blotting. Western blotting data presented are representative of those obtained in at least three separate experiments. HDW, Hedyotis diffusa Willd; ATO, arsenic trioxide; DR, death receptor.

Figure 5.

Effect of HDW on apoptosis-related proteins. WEHI-3 cells treated with HDW at 0.4, 0.8 and 1.6 mg/ml for 48 h. (A) The protein expression of PARP, and caspase-3, −8, and −9 was measured by western blotting. (B) The protein expression of Bcl-2, Bcl-xL and survivin was measured by western blotting. (C) The protein expression of Bak and Bid was also measured by western blotting. Western blot data presented are representative of those obtained in at least three separate experiments. HDW, Hedyotis diffusa Willd; PARP, poly adenosine diphosphate ribose polymerase; Bcl, B-cell lymphoma; xL, extra large; t-Bid, truncated Bid.

Figure 6.

Effect of HDW and/or ATO on apoptosis-related proteins. WEHI-3 cells were treated with 1.6 mg/ml HDW, 1.6 µM ATO or a combination of HDW and ATO for 48 h. (A) The protein expression of PARP, and caspase-3, −8, and −9 was measured by western blotting. (B) The protein expression of Bcl-2, Bcl-xL and survivin was also measured by western blotting, as well as the protein expression of (C) Bak and Bid. Western blot data presented are representative of those obtained in at least three separate experiments. HDW, Hedyotis diffusa Willd; ATO, arsenic trioxide; PARP, poly adenosine diphosphate ribose polymerase; Bcl, B-cell lymphoma; xL, extra large.