Induction of apoptosis of malignant gliomas cells by a prenylated chalcone

Abstract

Context: Malignant gliomas are the most commonly diagnosed brain tumors in adults. Chalcone and its derivatives have shown potential against glioblastoma and malignant gliomas.

Objective: The inhibitory activity of geranyl prenylated chalcone was investigated in four glioma cell lines: C6, U87 MB, CNS-1 and 13-06 MB. Cell death caused by the prenylated chalcone was determined to be necrosis or apoptosis.

Materials and methods: The inhibitory activity of geranyl prenylated chalcone with 5, 10, 15, 20, 25, 30 and 40 μg/ml (treatment time: 24, 48 and 72 h) was investigated in C6, U87 MB, CNS-1 and 13-06 MB. Cell cycle distribution, DNA fragmentation, chromatin condensation and protein expression were used as indicators of apoptosis. The migration ability of glioma cells with 30 μg/ml prenylated chalcone after 24 and 36 h incubation was also studied by the scratch wound assay.

Results: After 24 h, treatment with 20 μg/ml prenylated chalcone reduced the proliferation (approximately 50%) of all four glioma cell lines (half maximal inhibitory concentration (IC₅₀) = 20 μg/ml). Glioma cell death was verified by the fluorescence-activated cell sorter as prenylated chalcone-induced apoptosis. After running the analysis of protein expression, apoptotic activity induced by the prenylated chalcone was caspase independent for the C6 and U87 MB cell lines, but caspase dependent for the 13-06 MB and CNS-1 cell lines. In addition, prenylated chalcone treatment (30 μg/ml) resulted in the inhibition of glioma cell migration after 24 and 36 h treatment.

Discussion and conclusion: Because prenylated chalcone-induced apoptosis inhibited the proliferation and reduced the invasiveness of glioma cells, the prenylated chalcone has potential as a new chemotherapeutic reagent in the treatment of malignant gliomas. The ultimate goal was to develop a novel potential multi-therapy for treating gliomas.

• Brain tumor
• cell death
• cell migration
• proliferation

Introduction

Malignant gliomas, the most commonly diagnosed brain tumors, are one of the more lethal forms of brain cancer. These malignant astrocytic tumors are highly infiltrative and invasive (Wen & Kesari, 2008). They exhibit a high cell proliferation rate and an aggressive growth pattern (Ohgaki & Kleihues, 2005). An estimated 18 000 new cases of brain and central nervous system tumors are diagnosed each year (Kotliarov et al., 2006; Wen & Kesari, 2008). Approximately 13 000 people die annually of these diseases in the United States. The high recurrence and death rates are caused by tumor cell migration. Despite aggressive treatments combining surgery, radiation and chemotherapy, the median 1-year survival rate has remained essentially unchanged (Kim et al., 2008; Siegelin et al., 2008). The invasiveness of glioma limits the efficacy of surgery and other similar therapies (Chen et al., 2011; Nakada et al., 2007). Most treatments fail to extend patient life by more than 1 year because cells from the bulk tumor have already invaded normal brain tissue at the time of surgery (Demuth & Berens, 2004; Kurozumi et al., 2012). In some cases, more treatments and modalities are required to effectively treat malignant tumors.

Prenylated chalcones are intermediate products in the biosynthesis of flavonoids that have been considered as anticancer agents (Kanadaswami et al., 2005). Chalcones and their derivatives are generated from flavaones, flavones, isoflavones or flavonols (Okwu & Ukanwa, 2010). A geranyl prenylated chalcone named WJ9708011 has been semisynthesized from naturally occurring nymphaeol C (Huang et al., 2007). It has a common phenylbenzopyrone structure (Figure 1). The chemical structure of chalcones includes open-chain flavonoids where the two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl system. Prenylated chalcones have shown potential against glioblastoma and malignant glioma cells (Huang et al., 2007; Teng et al., 2011). Kamal et al. (2011) and Liu et al. (2012) concluded that prenylated chalcones inhibit the proliferation of breast cancer and mouse lymphoma cells.

Figure 1. The general chemical structure of a typical prenylated chalcone compound.

Apoptosis is a defined type of programmed cell death, differing from traditional necrotic cell death (Hotchkiss et al., 2009). Previous studies have demonstrated that induction of tumor cell apoptosis prevents the occurrence of cellular inflammatory responses (Haanen & Vermes, 1995; Kuwano & Hara, 2000; Savill, 1997). Therefore, apoptosis exhibits better cancer therapeutic effects than necrosis. Several characteristics of apoptotic cells, including DNA fragmentation, chromatin condensation and apoptotic bodies, are not observed in necrotic cells and thus have been used to identify the occurrence of apoptosis (Nagata, 2000). Although chalcone-induced apoptosis has been demonstrated in previous studies (Lee et al., 2011; Ramaiah et al., 2011), the apoptosis-inducing activities of chalcones have not been evaluated.

In this study, the inhibitory activity of geranyl prenylated chalcone was investigated in four glioma cell lines. Cell death caused by the prenylated chalcone was determined to be necrosis or apoptosis. Cell cycle distribution, DNA fragmentation, chromatin condensation and protein expression were used as indicators of apoptosis. The migration ability of glioma cells was also studied by the scratch wound assay. The ultimate goal was to develop a novel potential multi-therapy for treating gliomas.

Materials and methods

Cell culture preparation

Four glioma cell lines, including C6 (rat glioma cell), U87 MB (human malignant glioblastoma), CNS-1 (glioblastoma) and 13-06T MB (malignant glioma), were obtained from National Cheng Kung University (Tainan, Taiwan). Cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen^TM, Gibco, New York, NY) containing 10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin–neomycin (Invitrogen^TM, Gibco) in 75-cm³ flasks incubated at 37 °C in 5% CO₂-enriched air. Subcultures of confluent cells were prepared using 0.02% ethylenediaminetetraacetic acid (EDTA)/0.05% trypsin for culture in 96-well tissue culture plates at 37 °C in 5% CO₂-enriched air. EDTA and trypsin were purchased from Sigma-Aldrich (St. Louis, MO). Confluent monolayer culture was obtained after 1–2 d.

Cell viability analysis

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay. Glioma cells in the 96-well culture plates were treated with the prenylated chalcone (WJ970811, National Cheng Kung University, Tainan, Taiwan). Dimethyl sulfoxide (DMSO, Acros Organics, Fair Lawn, NJ) was used as the solvent. After 24, 48 and 72 h treatment, 0.5 mg/ml MTS was added and the cells were incubated for an additional 2 h at 37 °C. Prepared samples were analyzed using an enzyme-linked immunosorbent assay reader (Multiskan RC, Labsystems Inc., Franklin, MA). The absorbance of each sample was measured at a wavelength of 550 nm at 25 °C. Cell viability (%) was calculated by comparison between treated samples and the control unit.

Morphological analysis of cell apoptosis

Glioma cell apoptosis was determined by morphological analysis. The glioma cells treated with the prenylated chalcone (35 μg/ml, 48 h) were seeded on a slide and then placed in a 10-cm-diameter Petri dish. After 48-h incubation, the cells were washed three-times with cold phosphate-buffered saline (PBS), and dried for 12 h. The dried cells were observed using a fluorescence microscope (model no. Eclipse, E600; Nikon, Tokyo, Japan) after terminal deoxynucleotidyl transferase dUTP nick end labeling staining (Invitrogen^TM, Gibco).

Gel electrophoresis of DNA

Glioma cells treated with different concentrations of the prenylated chalcone (25, 30 and 35 μg/ml) were collected, washed two-times with PBS and lysed for 3 h at 56 °C in 100 ml of lysis buffer containing 50 mM Tris, 10 mM EDTA, 0.5% sodium sarcosinate and 1 mg/ml of proteinase K. DNA was extracted with phenol/chloroform/isoamyl alcohol. DNA samples were mixed with buffer containing 1% agarose and 0.025% (w/w) bromophenol blue (Sigma-Aldrich). The samples were loaded into a pre-solidified 2% agarose gel with 0.01 μg/ml ethidium bromide. The electrophoresis was run at 50 V for 45 min in 0.5% tris borate-EDTA (TBE) buffer, and results were visualized under an ultraviolet light.

Cell cycle distribution analysis

Glioma cells treated with different concentrations of the prenylated chalcone (25, 30 and 35 μg/ml) were incubated in six-well plates for 24 and 48 h. The cells were collected and washed with 1 ml of PBS. After adding 3 ml of pure ethanol, the mixture was stored at −30 °C for 12 h and centrifuged at 1500 rpm for 5 min. After washing with PBS, 1 μl of RNase A was added, and the mixture was incubated in a 37 °C water bath for 15–30 min. Then 500 μl of propidium iodide solution was added and protected from light for 10 min before running the analysis. Cells stained with propidium iodide located at sub-G1, which was defined as apoptotic.

Apoptotic cell analysis using a fluorescence-activated cell sorter

Glioma cells treated with 30 μg/ml prenylated chalcone were incubated in six-well plates for 24, 36 and 48 h. The cells were collected and washed with PBS. Apoptotic cells were detected by the Annexin V and propidium iodide staining method. The cells stained with Annexin V, located in the bottom right area, represented early apoptotic cells. Whereas the cells stained with both Annexin V and propidium iodide, located in the upper right area, represented late apoptotic cells.

Western blot analysis

Glioma cells treated with the prenylated chalcone were incubated for 48 h and disrupted in lysis buffer containing 1% Triton X-100, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM EDTA and 10 mM Tris-HCl (pH 7.4). Then, total cell extracts (30 μg) were mixed with 8% sodium dodecyl sulfate (SDS)-polyacrylamide minigels for poly ADP ribose polymerase (PARP) detection. In addition, total cell extracts (30 μg) were mixed with 10% SDS-polyacrylamide minigels for caspase-3, the Bcl-2 family and β-actin analyses. The extracts were then transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk at room temperature for 1 h and incubated with different primary antibodies for 12 h. After incubation was complete, these membranes were washed and incubated with secondary antibodies for 1 h. An enhanced chemiluminescence system was used for the visualization of the results.

Scratch wound assay

Glioma cells were plated in a six-well culture plate. After cells reached confluence, a 200 μl plastic pipette tip was moved quickly across the plate to create a scratch on the surface. The cells were washed two-times with PBS and incubated with 30 μg/ml prenylated chalcone. Images were captured after 24 and 36 h incubations.

Results and discussion

Cell viability analysis

The cytotoxic effect of the prenylated chalcone was determined by the treatment of four glioma cell lines. The control unit was rat primary fibroblast cells. Cell cultures were treated with the prenylated chalcone at various concentrations for 24, 48 and 72 h. No toxic effect was detected on the control unit cells treated with 25 μg/ml prenylated chalcone (data not shown), whereas cell numbers of the four glioma lines were reduced in all cases (Figure 2). This inhibition was dose-dependent. After 24 h, treatment with 20 μg/ml prenylated chalcone reduced the proliferation (approximately 50%) of all four glioma cell lines (half maximal inhibitory concentration (IC₅₀) = 20 μg/ml). Cell numbers further reduced at increased prenylated chalcone concentrations. The prenylated chalcone damaged cells and reduced proliferation with highest inhibitory activity against 13-06 MB cells (Figure 2). Zhang et al. (2010) reported that prenylated chalcones inhibit the proliferation of cancer cells. Champelovier et al. (2011) also reported that two compounds derived from chalcones have the ability to inhibit the proliferation of glioblastoma cells. Necrosis of cancer cells was one reason for the reduction in cell number. The other reason was apoptosis caused by information blocking inside cells. Both necrosis and apoptosis decreased the number of cancer cells. However, whether the inhibitory activity of the prenylated chalcone caused necrosis or apoptosis of glioma cells needed to be determined.

Figure 2. Effects of prenylated chalcone treatments on cell viability of C6, U87 MB, CNS-1 and 13-06 MB glioma cell lines. Data are the mean ± SD of triplicate analyses (n = 3). (*p < 0.05; **p < 0.01 versus control).

untreated,

5 μg/ml,

10 μg/ml,

15 μg/ml,

20 μg/ml,

25 μg/ml,

30 μg/ml and

40 μg/ml prenylated chalcone.

Cell cycle distribution analysis

In this study, the prenylated chalcone inhibited glioma cell growth by changing the cell shape and cell cycle phase. The prenylated chalcones influenced on the G1 cell cycle. Fluorescence-activated cell sorter (FACS) was used to evaluate changes in DNA that occurred inside glioma cells. FACS was also used for quantitative analysis. The glioma cells were treated with 25, 30 and 35 μg/ml of the prenylated chalcone for 24 and 48 h. Then the cells were stained with propidium iodide. The cell cycle phase of all four glioma cell lines increased in sub-G1 (Figure 3). The number of glioma cells in the sub-G1 cell cycle phase was higher at 24 h than at 48 h. This change provided an indication that cell apoptosis was a consequence of treatment with the prenylated chalcone.

Figure 3. Effect of prenylated chalcone on cell cycle distribution of glioma cells. Data (% of cell in the indicated phase) are the mean ± SD of triplicate analysis (n = 3).

untreated,

25 μg/ml,

30 μg/ml and

35 μg/ml prenylated chalcone.

Apoptotic cell analysis

The numbers of hypodiploid (apoptotic) and diploid (non-apoptotic) cells were also analyzed using FACS to evaluate the apoptotic ratio of glioma cells treated with the prenylated chalcone (Figure 4A). Live annexin V(−)/PI(−) cells were represented in the lower left quadrant (Q3) of the scatter plot. Early and late apoptotic cells labeled annexin V(+)/PI(−) or V(+)/PI(+) were represented in right quadrants (Q2 and Q4, respectively). Glioma cells were treated with 30 μg/ml prenylated chalcone and incubated for 24, 36 and 48 h to verify if reduced cell viability was caused by apoptosis (Figure 4B). The glioma cells in the Q2 and Q4 quadrants increased with incubation time, verifying prenylated chalcone-induced glioma cell death as apoptosis. The apoptosis-inducing action of polyphenols involved several mechanisms including p53 or B-cell lymphoma 2 (Bcl-2)/Bax proteins (Cho et al., 2012). Thus, it was important to understand why the prenylated chalcone caused glioma cell apoptosis.

Figure 4. Fluorescence-activated cell sorter (FACS) analyses of apoptotic glioma cells. (A) Glioma cells treated with 15 and 30 μg/ml prenylated chalcone for 24 h. (B) Glioma cells treated with 30 μg/ml prenylated chalcone for 24, 36 and 48 h.

Condensation of chromatin DNA was a typical nuclear morphology when cells underwent apoptosis (Kerr et al., 1972). During apoptosis, the phase of chromatin changed from an active heterogeneous network to a highly condensed inert form. Chromatin DNA was condensed into several small bright granules (Figure 5A). The chromatin was fragmented and packed into apoptotic bodies. Chromatin condensation (Figure 5A) and DNA fragmentation (Figure 5B) within the nuclei of dying cells were the most recognizable evidence of apoptosis (Wyllie, 1980; Wyllie et al., 1984).

Figure 5. Apoptosis induced by prenylated chalcone treatment in glioma cells. (A) Chromatin condensation caused by 35 μg/ml prenylated chalcone at 24 h. (B) DNA fragmentation caused by 25, 30 and 35 μg/ml prenylated chalcone at 48 h. Marker: a 100-bp DNA ladder (left lane).

Activation of apoptosis-related protein in glioma cells

Analysis of protein expression is a good indicator for the determination of the many pathways involved in cell apoptosis. Effects of the prenylated chalcone on protein expression are shown in Figure 6. Glioma cells were treated with 25, 30 and 35 μg/ml of the prenylated chalcone. Protein expression of caspase3 cleavage and PARP cleavage were upregulated, especially in CNS-1 and U87 MB cells treated with 30 μg/ml prenylated chalcone. The prenylated chalcone increased expression of BAD and BAX pro-apoptosis proteins in the CNS-1 and 13-06 MB cells. Meanwhile, prenylated chalcone treatment reduced Bcl-2 and Bcl-XL pro-/anti-apoptosis proteins in the C6 and U87 MB cells. Thus, the prenylated chalcone induced apoptosis in the four glioma cell lines. In this study, apoptotic activity induced by the prenylated chalcone was caspase independent for the C6 and U87 MB cell lines, but caspase dependent for the 13-06 MB and CNS-1 cell lines. Why apoptosis of 13-06 MB and CNS-1 cell lines was caspase dependent requires further study.

Figure 6. Effects of prenylated chalcone on apoptotic protein expression using western blotting. (A) Anti-proapoptosis (Bcl-2 and Bcl-XL) and anti-apoptosis (Bad and Bax) (B) PARP and anti-caspase 3. Glioma cells were treated with 25, 30 and 35 μg/ml prenylated chalcone.

Glioma cell migration study using the scratch wound assay

Proliferation and invasive behavior are important characteristics of cancer cells and good indicators of malignancy. Invasion is usually recognized as the main reason for the high recurrence and death rates associated with glioma (Kurozumi et al., 2012). In general, the development of an anti-cancer reagent targets against the proliferation or invasive behavior of cancer cells. In this study, the prenylated chalcone inhibited not only proliferation but also migration of glioma cells. Dorn et al. (2010) reported that prenylated chalcones show good inhibitory activity against the proliferation and migration of hepatocytes in vitro. Because prenylated chalcones have the potential to be a promising therapeutic agent (Dorn et al., 2010), it is necessary to study their influence of the invasiveness of glioma cells. Figure 7 shows the effect of prenylated chalcone treatment on glioma cell migration using the scratch wound assay. The shape of glioma cells was altered by prenylated chalcone treatment. The cells lost their spindle and became round in shape, and their migratory ability was limited, whereas untreated cells moved freely. Prenylated chalcone treatment (30 μg/ml) resulted in the inhibition of glioma cell migration after 24 and 36 h treatment.

Figure 7. Effect of prenylated chalcone on glioma cell migration using the scratch wound assay. Glioma cells were incubated with 30 μg/ml prenylated chalcone for 24 and 36 h. (A) C6, (B) U87 MB, (C) CNS-1 and (D) 13-06 MB.

Conclusions

Malignant brain tumors (gliomas) are rarely cured by surgery, radiotherapy or chemotherapy alone. A combination of treatments is usually necessary to effectively treat these malignant gliomas. Geranyl prenylated chalcone successfully reduced the proliferation of four glioma cell lines: C6, U87 MB, CNS-1 and 13-06 MB. The death of gliomas cells induced by the prenylated chalcone was verified as apoptosis. Prenylated chalcone-induced apoptosis inhibited the proliferation of glioma cells. The prenylated chalcone also reduced the invasiveness of gliomas cells. Thus, the prenylated chalcone has the potential as a new chemotherapeutic reagent in the treatment of malignant gliomas.

Declaration of interest

The authors report no declarations of interest.