Novel Structurally Related Flavones Augment Cell Death Induced by rhsTRAIL

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) was identified as a powerful activator of apoptosis in tumor cells and one of the most promising candidates for cancer therapy with no toxicity against normal tissues. However, many tumor cells are resistant to TRAIL-induced apoptosis. The aim of this work was to analyze the improvement of the anticancer effect of rhsTRAIL (recombinant human soluble TRAIL) by nine flavones: 5-Hydroxyflavone, 6-Hydroxyflavone, 7-Hydroxyflavone and their new synthetic derivatives 5-acetoxyflavone, 5-butyryloxyflavone, 6-acetoxyflavone, 6-butyryloxyflavone, 7-acetoxyflavone and 7-butyryloxyflavone. We examined the cytotoxic and apoptotic effects of rhsTRAIL enhanced by novel structurally-related flavones on SW480 and SW620 colon cancer cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test, the lactate dehydrogenase assay and annexin V-FITC fluorescence staining. We observed a slight difference in the activities of the flavones that was dependent on their chemical structure. Our study indicates that all nine flavones significantly augment cell death by rhsTRAIL (cytotoxicity range 36.8 ± 1.7%–91.4 ± 1.7%; apoptosis increase of 33.0 ± 0.7%–78.5 ± 0.9%). Our study demonstrates the potential use of tested flavones in TRAIL-based anticancer therapy and prevention.

Because biochemical action depends on the individual flavonoid structure, each compound should be evaluated systematically to assess its individual biological potency. Accordingly, the anticancer effects of flavone analogs alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were analyzed.
Numerous studies have reported interesting properties of flavonoids in anticancer strategies, with notable pleiotropic influences on immune and cancer cells [6]. Flavones exhibit chemopreventive and antitumor actions by interfering with apoptosis or proliferation signaling and regulating the immune system [1,7,8]. TRAIL, a TNF (tumor necrosis factor) superfamily member, is considered to be an endogenous anticancer agent because of its selective cytotoxicity against tumor cells compared to normal primary cells [9,10]. This death ligand is expressed on T lymphocytes, natural killer cells, neutrophils, monocytes and macrophages [7,11]. Membrane-bound TRAIL can be cleaved from the cell surface into a soluble secreted form. Whether soluble or expressed on immune cells, TRAIL molecules play an important role in the surveillance and elimination of tumors [12,13]. The growing interest in TRAIL-based interventions has led to the development of recombinant human TRAIL (rhTRAIL) as a promising therapy for different types of human cancer [14]. The preliminary results of phase I and II clinical trials on rhTRAIL in patients with advanced neoplastic diseases indicate that the direction of these studies is proper. However, some types of cancer cells are resistant to TRAIL-mediated apoptosis [15,16]. The induction of cancer cell-specific apoptosis via enhanced TRAIL signaling has become an important focus of cancer research [17,18]. Most commonly-used standard chemotherapeutic agents including gemcitabine, irinotecan, doxorubicin, 5-fluorouracil and cisplatin, as well as natural or new synthetic compounds have been shown to sensitize cancer cells to TRAIL-mediated apoptosis [7,19]. Various studies confirm that flavones also augment the antitumor activity of TRAIL and overcome TRAIL resistance in cancer cells [7] and suggest a significant role for various natural flavones in anticancer immune effector mechanisms [7,20].
In the present study, we examined the cytotoxic and apoptotic effects of recombinant human soluble TRAIL (rhsTRAIL) in combination with nine natural and synthetic flavones: 5-Hydroxyflavone (5-HF) and its derivatives, 5-acetoxyflavone (5-AF) and 5-butyryloxyflavone (5-BF), 6-Hydroxyflavone (6-HF) and its derivatives, 6-acetoxyflavone (6-AF) and 6-butyryloxyflavone (6-BF), and 7-Hydroxyflavone (7-HF) and its derivatives, 7-acetoxyflavone (7-AF) and 7-butyryloxyflavone (7-BF) (Figure 1) in SW480 and SW620 colon cancer cells. cells compared to normal primary cells [9,10]. This death ligand is expressed on T lymphocytes, natural killer cells, neutrophils, monocytes and macrophages [7,11]. Membrane-bound TRAIL can be cleaved from the cell surface into a soluble secreted form. Whether soluble or expressed on immune cells, TRAIL molecules play an important role in the surveillance and elimination of tumors [12,13]. The growing interest in TRAIL-based interventions has led to the development of recombinant human TRAIL (rhTRAIL) as a promising therapy for different types of human cancer [14]. The preliminary results of phase I and II clinical trials on rhTRAIL in patients with advanced neoplastic diseases indicate that the direction of these studies is proper. However, some types of cancer cells are resistant to TRAIL-mediated apoptosis [15,16]. The induction of cancer cell-specific apoptosis via enhanced TRAIL signaling has become an important focus of cancer research [17,18]. Most commonly-used standard chemotherapeutic agents including gemcitabine, irinotecan, doxorubicin, 5-fluorouracil and cisplatin, as well as natural or new synthetic compounds have been shown to sensitize cancer cells to TRAIL-mediated apoptosis [7,19]. Various studies confirm that flavones also augment the antitumor activity of TRAIL and overcome TRAIL resistance in cancer cells [7] and suggest a significant role for various natural flavones in anticancer immune effector mechanisms [7,20].

Cytotoxic and Apoptotic Effects of Flavones in Colon Cancer Cells
Various in vitro and in vivo studies confirm the anticancer activity of flavones [1,[21][22][23]. In addition to two natural flavones (5-HF (also known as primuletin) and 7-HF), another seven analogs (5-AF, 5-BF, 6-HF, 6-AF, 6-BF, 7-AF, 7-BF) are synthetic compounds. First, we examined the cytotoxicity and apoptosis induced by nine flavones, 5-HF, 6-HF, 7-HF and their new synthetic derivatives (5-AF, 5-BF, 6-AF, 6-BF, 7-AF, 7-BF) at concentrations of 50 µM and 100 µM in SW480 and SW620 colon cancer cells (Figures 2 and 3). Flavones cause their cytotoxic effect in colon cancer cells via the apoptotic route. The necrotic cell death percentage of cancer cells examined by the lactate dehydrogenase (LDH) assay and flow cytometry with propidium iodide was near 0%. In our work, we describe for the first time the enhancement of rhsTRAIL-mediated apoptosis in cancer cells by nine flavones: 5-HF, 6-HF, 7-HF and their new synthetic derivatives with an acetyl or a butyryl group.

Cytotoxic and Apoptotic Effects of Flavones in Colon Cancer Cells
Various in vitro and in vivo studies confirm the anticancer activity of flavones [1,[21][22][23]. In addition to two natural flavones (5-HF (also known as primuletin) and 7-HF), another seven analogs (5-AF, 5-BF, 6-HF, 6-AF, 6-BF, 7-AF, 7-BF) are synthetic compounds. First, we examined the cytotoxicity and apoptosis induced by nine flavones, 5-HF, 6-HF, 7-HF and their new synthetic derivatives (5-AF, 5-BF, 6-AF, 6-BF, 7-AF, 7-BF) at concentrations of 50 μM and 100 μM in SW480 and SW620 colon cancer cells (Figures 2 and 3). Flavones cause their cytotoxic effect in colon cancer cells via the apoptotic route. The necrotic cell death percentage of cancer cells examined by the lactate dehydrogenase (LDH) assay and flow cytometry with propidium iodide was near 0%.  Cytotoxic and apoptotic effects of rhsTRAIL on SW480 and SW620 colon cancer cells. Cells were incubated with 25-100 ng/mL TRAIL for 48 h. The values represent the mean ± SD of three independent experiments (n = 3). (A) Cytotoxic activity of rhsTRAIL against colon cancer cells. The percentage of cell death was measured using the MTT cytotoxicity assay (*** p < 0.001 compared to control without rhsTRAIL); (B) Apoptotic activity of rhsTRAIL against colon cancer cells. Apoptotic cell death was detected by flow cytometry using annexin V-FITC staining (*** p < 0.001 compared to control without rhsTRAIL). The percentage of cell death was measured using the MTT cytotoxicity assay (*** p < 0.001 compared to control without rhsTRAIL); (B) Apoptotic activity of rhsTRAIL against colon cancer cells. Apoptotic cell death was detected by flow cytometry using annexin V-FITC staining (*** p < 0.001 compared to control without rhsTRAIL).
The activity of the flavones was dependent on the dose and structure of the compound and on the tested cell line, with 7-HF and its two analogs at 50 µM and 100 µM possessing the strongest anticancer properties (Supplementary Figures S1 and S2). The obtained data indicate higher activity of the tested flavones against SW620 than SW480.
The obtained results suggest that a hydroxyl group located at the C6 or C7 position, an acetoxyl group located at the C6 or C7 position (and also C5 position for SW620) and a butyryl group located at the position C5, or C6, or C7 determines the strength of the cytotoxic and apoptotic effects of the compounds against colon cancer cells. We observed differences in the sensitivity of the malignant cell lines in our study; in contrast to SW480 cells, SW620 cells were more susceptible to the anticancer activity of flavones.
The obtained results suggest that a hydroxyl group located at the C6 or C7 position, an acetoxyl group located at the C6 or C7 position (and also C5 position for SW620) and a butyryl group located at the position C5, or C6, or C7 determines the strength of the cytotoxic and apoptotic effects of the compounds against colon cancer cells. We observed differences in the sensitivity of the malignant cell lines in our study; in contrast to SW480 cells, SW620 cells were more susceptible to the anticancer activity of flavones.

Cytotoxic and Apoptotic Effects of TRAIL in Combination with Flavones in Colon Cancer Cells
The rhsTRAIL used in our study is a soluble protein based on a natural endogenous ligand [14,24]. We first tested the anticancer effect of rhsTRAIL on both colon cancer cell lines ( Figure 4). The cell death induced by 25-100 ng/mL TRAIL in the SW480 cell line reached 20.8 ± 0.6%-28.7 ± 1.3% and rhsTRAIL at concentrations of 50-100 ng/mL caused 12.9 ± 1.0%-18.8 ± 1.0% cell death in the SW620 cell line. The necrotic cell death percentage of cancer cells revealed by an LDH assay and flow cytometry with propidium iodide was near 0%. rhsTRAIL at the same concentration triggered apoptosis in 26.2 ± 0.7%-29.8 ± 0.9% of SW480 cells and in 12.9 ± 1.0%-16.0 ± 1.1% of SW620 cells. rhsTRAIL at concentrations of 200 ng/mL or higher did not significantly increase this anticancer effect on the cancer cells (data not shown).  We confirmed that both colon cancer cell lines are resistant to rhsTRAIL-induced apoptosis. The SW480 cell line was more susceptible to the anticancer activity of rhsTRAIL than the SW620 cell line. Therefore, in further studies, we used rhsTRAIL at 25 ng/mL and 50 ng/mL for the SW480 cells and 50 ng/mL and 100 ng/mL for the SW620 cells.
The TRAIL-mediated apoptotic pathway is a potential target for phenolics and polyphenols [7,25]. Next, the cytotoxic and apoptotic activities of rhsTRAIL in combination with nine flavone analogs at 50 μM and 100 μM on colon cancer cells were investigated. All tested flavones overcame TRAIL We confirmed that both colon cancer cell lines are resistant to rhsTRAIL-induced apoptosis. The SW480 cell line was more susceptible to the anticancer activity of rhsTRAIL than the SW620 cell line. Therefore, in further studies, we used rhsTRAIL at 25 ng/mL and 50 ng/mL for the SW480 cells and 50 ng/mL and 100 ng/mL for the SW620 cells.
The TRAIL-mediated apoptotic pathway is a potential target for phenolics and polyphenols [7,25]. Next, the cytotoxic and apoptotic activities of rhsTRAIL in combination with nine flavone analogs at 50 µM and 100 µM on colon cancer cells were investigated. All tested flavones overcame TRAIL resistance in the SW480 and SW620 cells. The combined anticancer action of TRAIL and the tested flavones was dependent on the dose of ligand, the dose and structure of the compound and the type of cell line. The compounds significantly augmented the cytotoxic and apoptotic effects of TRAIL (cytotoxicity increased to 45.1 ± 1.4%-91.4 ± 1.7% in SW480 cells and 36.8 ± 1.7%-90.0 ± 1.1% in SW620 cells; apoptosis increased to 37.1 ± 0.9%-74.4 ± 1.1% in SW480 cells and 33.0 ± 0.7%-78.5 ± 0.9% in SW620 cells).
The co-treatment of rhsTRAIL and the compounds with an acetyl or a butyryl group at position 7 (7-AF, 7-BF) induced significant and the strongest cytotoxicity in SW480 and SW620 cells compared to 7-HF; 7-AF and 7-BF significantly augmented rhsTRAIL-induced cytotoxicity in both colon cancer cell lines compared to 7-HF. 5-AF and 5-BF combined with rhsTRAIL used against SW620 cells and 6-AF and 6-BF combined with rhsTRAIL used against SW480 caused also the strongest cytotoxic activity, as their primary analogs (5-HF and 6-HF, respectively).
We observed slight differences between the cytotoxic and apoptotic effect; the cytotoxicity after application of rhsTRAIL and flavones was higher as a result of apoptosis. This indicated that except apoptosis, also other types of cell death could be involved in the cytotoxic effect.
We proved that all tested flavone analogs showed a significant anticancer effect. The compounds with a hydroxyl group located at the C6 or C7 position, an acetoxyl group located at the C6 or C7 position (and also the C5 position for SW620) and a butyryl group located at the position C5, or C6, or C7 demonstrated the strong cytotoxic and apoptotic activities against colon cancer cells. In addition, we noticed differences in the sensitivity of the tested colon cell lines; SW620 cells were more susceptible to the anticancer effect of flavones compared to SW480 cells.
The death ligand TRAIL is a powerful inducer of apoptosis in cancer cells [42]. Endogenous TRAIL expressed on the surface of immune cells or cleaved into a soluble, secreted form plays an important role in the surveillance and defense against malignant tumors [12,13,43,44]. In recent years, numerous exogenous forms of TRAIL have been developed based on the structure and biological activities of the natural ligand. Both pre-clinical and clinical studies with recombinant human soluble TRAIL (rhsTRAIL) have shown a remarkable anticancer effect in a wide range of tumor types [14,15]. However, some cancer cells are resistant to TRAIL-mediated apoptosis [45,46]. The expression of death receptors and proapoptotic or antiapoptotic proteins in cancer cells is involved in TRAIL resistance [15,16,43]. We tested the effect of rhsTRAIL on SW480 and SW620 cells and confirmed that both colon cancer cell lines are resistant to TRAIL-induced apoptosis [45,46]. The SW480 cell line was more susceptible to the anticancer activity of rhsTRAIL than the SW620 cell line.
rhsTRAIL-resistant cancer cells can be sensitized to rhsTRAIL-induced apoptosis with various natural and synthetic flavones [7,47,48]. Next, we examined for the first time the cytotoxic and apoptotic activities of rhsTRAIL in combination with nine flavone analogs on colon cancer cells. The co-treatment effect of TRAIL and flavones was dependent on the concentration of the ligand, on the concentration and structure of the compound and on the type of cell line. All nine flavones significantly augment the anticancer activity of rhsTRAIL. The flavones with a hydroxyl or an acetyl or a butyryl group at position 7 (7-HF, 7-AF, 7-BF) in combination with rhsTRAIL exhibited the highest effect against both colon cancer cell lines. The flavone derivatives with an acetyl or a butyryl group at position 6 (6-AF, 6-BF) and 5-HF in combination with rhsTRAIL exhibited the highest effect against SW480 cells. In contrast, the flavone analogs with an acetyl or a butyryl group at position 5 (5-AF, 5-BF) in combination with TRAIL showed the strongest effect against SW620 cells.
TRAIL is considered as an effective inducer of death in cancer cells under clinical investigation. The enhancement of the TRAIL-mediated apoptosis by novel compounds possibly represents a promising strategy.

General Procedure for the Esterification of 5-HF, 6-HF and 7-HF
To 0.25 mmol of 5-HF, 6-HF or 7-HF dissolved in 5 mL of tetrahydrofuran (THF), 0.62 mmol of pyridine and 0.58 mmol of acetyl chloride (butyryl chloride) were added. The reaction mixture was stirred with a magnetic stirrer at room temperature for 30 min (the progress of the reaction was monitored by TLC). When the substrate was fully consumed, 5 mL of ethyl acetate were added, and the reaction mixture was washed with 0.5 M HCl until the solution became slightly acidic (pH = 5). The organic layer was then separated, and the aqueous layer was additionally extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with brine until neutral and dried over anhydrous MgSO4. The esters were separated by column chromatography (SiO2) (Scheme 1).

Cell Culture
The experiments were performed on SW480 and SW620 human colon cancer cells obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). The SW480 and SW620 cells were grown in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 • C in a humidified atmosphere of 100% air [45,46]. The reagents for the cell culture were purchased from ATCC or PAA Laboratories (Pasching, Austria).

Lactate Dehydrogenase Release Assay
Lactate dehydrogenase (LDH) is a stable cytosolic enzyme released from necrotic cells upon membrane damage. The measurement of LDH activity was performed using a commercial cytotoxicity assay kit (Roche Diagnostics GmbH, Mannheim, Germany). LDH is detected in culture supernatants using a coupled enzymatic assay, resulting in the conversion of a tetrazolium salt into a red formazan product. SW480 and SW620 cells were treated with various concentrations of flavones (50-100 µM dissolved in DMF) and/or TRAIL (25-200 ng/mL) for the indicated period of time. The sample solution (supernatant) was removed, and the LDH released from the cells was measured in the culture medium. The maximal release of LDH (positive control) was obtained after treating cells with 1% Triton X-100 for 10 min at room temperature. The spectrophotometric absorbance was measured at 490 nm using a microplate reader (EL x800, Bio-Tek Instruments Inc., Winooski, VT, USA) [52,66]. The necrotic percentage was expressed using the formula: (sample value/maximal release) × 100%.

Determination of Apoptosis by Flow Cytometry with Annexin V-FITC Staining
Apoptosis was determined by flow cytometry using the Apoptest-FITC Kit with annexin V (Dako, Glostrup, Denmark). SW480 cells (5 × 10 5 /mL) and SW620 cells (2.5 × 10 5 /mL) were seeded in 24-well plates for 24 h prior to experimentation and then exposed to flavones (50-100 µM dissolved in DMF) and/or TRAIL (25-200 ng/mL) for 48 h. After incubation, the cells were washed twice with phosphate-buffered saline solution (PBS) and resuspended in 500 µL of binding buffer. The cell suspension (290 µL) was then incubated with 5 µL of annexin V-FITC and 5 µL of propidium iodide for 10 min at room temperature in the dark. The population of annexin V-positive cells was evaluated by flow cytometry (BD LSR II Analyzer, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) [49,66].

Statistical Analysis
The results are expressed as the means ± SD obtained from three or four separate experiments performed in duplicate (n = 6 or n = 8) or quadruplicate (n = 12). Statistical significance was evaluated using Student's t test. p < 0.05 was considered significant.

Conclusions
In summary, we synthesized a series of novel flavone derivatives and examined their anticancer effect alone and in combination with TRAIL. The compounds displayed low anticancer activity, but in combination with rhsTRAIL, the effect was enhanced. Our study demonstrates the potential use of tested flavones in TRAIL-based anticancer therapy and prevention. However, further in vitro and in vivo investigations are required to explain the mechanism of action of the flavone analogs.