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. 2013:2013:434709.
doi: 10.1155/2013/434709. Epub 2013 Feb 28.

5,7-Dihydroxyflavone Enhances the Apoptosis-Inducing Potential of TRAIL in Human Tumor Cells via Regulation of Apoptosis-Related Proteins

Zhenzhen Zhang  1 , Tingmei Ye, Xueting Cai, Jie Yang, Wuguang Lu, Chunping Hu, Zhigang Wang, Xiaoning Wang, Peng Cao
Affiliations

5,7-Dihydroxyflavone Enhances the Apoptosis-Inducing Potential of TRAIL in Human Tumor Cells via Regulation of Apoptosis-Related Proteins

Zhenzhen Zhang et al. Evid Based Complement Alternat Med. 2013.

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy.

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Figures

Figure 1
Figure 1
Chemical structure of 5,7-dihydroxyflavone (C15H10O4, CAS No: 480-40-0, Mol. Wt.: 254.24).
Figure 2
Figure 2
Interactive effects of 5,7-dihydroxyflavone and TRAIL on cell viability. (A), HepG2 cells were treated with various concentrations of 5,7-dihydroxyflavone and/or TRAIL. Cell viability was measured at the end of 72 h by MTT assay. Values marked with asterisk(s) are significantly different (*P < 0.05, **P < 0.01) from control. Data are mean ± SD. (B), HepG2 cells were treated with 5,7-dihydroxyflavone and/or TRAIL for indicated lengths of time. Cell viability was measured by MTT assay. Values marked with asterisk(s) are significantly different (*P < 0.05, **P < 0.01) from control. Data are mean ± SD. (C), light micrograph of HepG2 cells treated with 5,7-dihydroxyflavone and/or TRAIL for 24 h (×200). (a, control; b, 6 nM TRAIL; c, 20 μM 5,7-dihydroxyflavone; d, 60 nM TRAIL; e, 6 nM TRAIL + 20 μM 5,7-dihydroxyflavone). (D), Jurkat cells were treated with 5,7-dihydroxyflavone and/or TRAIL for 48 h and cell viability was measured by MTT assay. Values marked with asterisk(s) are significantly different (*P < 0.05, **P < 0.01) from control. Data are mean ± SD. (E), Hela cells were treated with 5,7-dihydroxyflavone and/or TRAIL for 72 h and cell viability was measured by MTT assay. Values marked with asterisk(s) are significantly different (*P < 0.05, **P < 0.01) from control. Data are mean ± SD. (F), L-O2 cells were treated with various concentrations of 5,7-dihydroxyflavone and/or TRAIL. Cell viability was measured by MTT assay. Data are mean ± SD.
Figure 3
Figure 3
5,7-Dihydroxyflavone sensitized tumor cells to TRAIL-induced apoptosis. (A) HepG2 cells were treated with 5,7-dihydroxyflavone and/or TRAIL for 24 h. Apoptosis was assessed by propidium iodide DNA staining and flow cytometry. (B) HepG2, Jurkat or HeLa cells were treated with 5,7-dihydroxyflavone and/or TRAIL for 24 h. Apoptosis was assessed by hoechst33258 staining (×200) (HepG2 cells: a, control; b, 6 nM TRAIL; c, 20 μM 5,7-dihydroxyflavone; d, 60 nM TRAIL; e, 6 nM TRAIL + 20 μM 5,7-dihydroxyflavone; Jurkat and HeLa cells: a, control; b, 6 nM TRAIL; c, 20 μM 5,7-dihydroxyflavone; d, 6 nM TRAIL + 20 μM 5,7-dihydroxyflavone). (C) HepG2, Jurkat or HeLa cells were treated with 5,7-dihydroxyflavone and/or TRAIL for 24 h. Cell lysates were separated by SDS-PAGE, then procaspase-9, procaspase-3, and PARP proteins were detected by Western blot analysis. GADPH was used as loading control.
Figure 4
Figure 4
Influence of 5,7-dihydroxyflavone on DR4 and DR5 expressions. HepG2 cells were treated with or without 5,7-dihydroxyflavone for 24 h and then harvested for analysis of cell surface DR4 and DR5 by immunofluorescent staining and subsequent flow cytometry. Filled purple peaks, cells stained with a match isotype control; green line, untreated control; pink line, 20 μmol/L 5,7-dihydroxyflavone; blue line, 40 μmol/L 5,7-dihydroxyflavone.
Figure 5
Figure 5
Influence of 5,7-dihydroxyflavone on the expression of c-FLIP. HepG2 cells were treated with the indicated concentrations of 5,7-dihydroxyflavone for 24 h. Western blotting was performed to determine the protein levels of c-FLIP. GADPH was used as loading control.
Figure 6
Figure 6
Influence of 5,7-dihydroxyflavone on the expression of Bcl-2, Bax, Bcl-XL, and Mcl-1. (a) HepG2 cells were treated with the indicated concentrations of 5,7-dihydroxyflavone for 24 h. Western blotting was performed to determine the protein levels of Bcl-2, Bax, Bcl-XL, and Mcl-1. GADPH was used as loading control. (b)–(d) the relative densities of these proteins were determined following densitometric measurements of the specific protein bands and normalization against the value of GAPDH. The asterisk indicates a significant difference (*P < 0.05, **P < 0.01) compared with control. Data are mean ± SD.
Figure 7
Figure 7
Influence of 5,7-dihydroxyflavone on the expression of c-IAP1, c-IAP2, XIAP, and Survivin. (a) HepG2 cells were treated with the indicated concentrations of 5,7-dihydroxyflavone for 24 h. Western blotting was performed to determine the protein levels of c-IAP1, c-IAP2, XIAP, and Survivin. GADPH was used as loading control. (b)–(e) the relative densities of these proteins were determined following densitometric measurements of the specific protein bands and normalization against the value of GAPDH. The asterisk indicates a significant difference (**P < 0.01) compared with control. Data are mean ± SD.
Figure 8
Figure 8
Influence of 5,7-dihydroxyflavone on the activation of Akt, STAT3, and MAPK. (a) HepG2 cells were treated with the indicated concentrations of 5,7-dihydroxyflavone for 24 h. Western blotting was performed to determine the phosphorylation levels of Akt, STAT3, and mitogen-activated protein kinase (MAPK). GADPH was used as loading control. (b, c), the relative densities of phospho-Akt and phosphor-STAT3 were determined following densitometric measurements of the specific protein bands and normalization against the value of total Akt and STAT3, respectively. The asterisk indicates a significant difference (**P < 0.01) compared with control. Data are mean ± SD.
Figure 9
Figure 9
5,7-Dihydroxyflavone synergized with TRAIL in the treatment of hepatocarcinoma xenografts. HepG2 cells were implanted into athymic mice subcutaneously. When the average tumor volume reached about 150 mm3, mice (N = 9) were treated for 28 days with vehicle control, 5,7-dihydroxyflavone (30 mg/kg/d) by oral gavage, TRAIL (10 mg/kg/d) i.p., and 5,7-dihydroxyflavone (30 mg/kg/d) by oral gavage + TRAIL(10 mg/kg/d) i.p.. (a) and (d) on day 29, tumors were isolated and weighed. (b) the tumor growth was monitored twice a week. (c) animal weights were recorded during the 28-day treatment. The asterisk indicates a significant difference (*P < 0.05, **P < 0.01) compared with control. Data are mean ± SD.
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