Dichloromethane fraction from Gardenia jasminoides: DNA topoisomerase 1 inhibition and oral cancer cell death induction

Abstract

Context: A growing body of evidence shows that compounds of plant origin have the ability to prevent cancer. The fruit of gardenia, Gardenia jasminoides Ellis (Rubiaceae), has long been used as a food additive and herbal medicine, and its pharmacological actions, such as protective activity against oxidative damage, cytotoxic effect, and anti-inflammatory and anti-tumor activity, have already been reported.

Objective: The purpose of the present study was to investigate the presence of DNA topoisomerase 1 inhibitor in various solvent fractions of Gardenia extract and examine the induction of oral cancer cell death upon treatment with Gardenia extract.

Materials and methods: The methanol extract of Gardenia was partitioned with n-hexane, dichloromethane, ethyl acetate, n-butanol, and water.

Results: In the DNA topoisomerase 1 assay, n-hexane and dichloromethane fractions inhibited topoisomerase 1 and led to a decrease in the cell viability of KB cells. The dichloromethane fraction (0.1 mg/mL) also showed 77% inhibition of cell viability in KB cells compared with HaCaT cells. Treatment with dichloromethane fraction led to apoptotic cell death as evidenced by flow cytometric analysis and morphological changes. In addition, treatment with Gardenia extract dichloromethane fraction led to the partial increase of caspase-3, caspase-8 and caspase-9 activities and the cleavage of poly (ADP-ribose) polymerase.

Conclusion: Taken together, these results suggest that the dichloromethane fraction from Gardenia extract induces apoptotic cell death by DNA topoisomerase 1 inhibition in KB cells. These findings suggest the possibility that Gardenia extract could be developed as an anticancer modality.

Keywords::

• Gardenia jasminoides Ellis
• oral cancer
• DNA topoisomerase 1

Introduction

Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral cavity and represents about 90% of all oral malignancies. As many as 30–50% of patients with oral malignancy develop local or regional recurrence, and an increasing number of patients develop distant metastases (Myers et al., 2001). Another 10–40% of patients develop second primary tumors of the aerodigestive tract as a result of field cancerization (Ferguson, 1994; Hong et al., 1990). Despite standard treatment strategies which involve surgery, radiotherapy or chemotherapy, the survival of patients with this cancer remains poor. An alternative method is needed to achieve a better prognosis for patients of oral SCC. A number of recent studies have focused on anticarcinogenic, antimutagenic, or chemopreventive activities of phytochemicals, particularly those included in human diet (Ferguson, 1994; Stavric, 1994).

DNA topoisomerase 1 (TOP1)-targeted drugs represent a novel class of anticancer agents that exert potent efficacy toward a variety of solid tumors (Chen et al., 2004). DNA topoisomerase 1 plays an essential role in DNA replication and DNA transcription. Some of the most commonly employed antitumor drugs attack topoisomerase, taking advantage of its key role in cellular information flow. Compounds based on the plant toxin camptothecin attack type 1 topoisomerase. The drugs bind to the DNA-topoisomerase complex after DNA-strand incisions have been made, blocking the enzymatic reaction that reseals the DNA. The result is breakage of the DNA and interference with cell cycle progression, resulting in cancer cell death (Goodsell, 2002; Stravopodis et al., 2009).

The fruit of gardenia [(Gardenia jasminoides Ellis (Rubiaceae)] has been used in traditional oriental medicine. As such, its use has been reported for the treatment of inflammation, jaundice, headache, edema, fever, hepatic disorders, and hypertension (Park et al., 2003), and pigments from the fruit are used as food colorants in Asian countries such as Korea, China and Taiwan. Gardenia fruit contains iridoid glycosides and crocin as major compounds (Machida et al., 2000; Ozaki et al., 2002), and these components exhibit antioxidant, cytotoxic, antitumor, and antihyperlipidemic effects (Hsu et al., 1999; Lee et al., 2005; Mathews-Roth, 1982; Tseng et al., 1995). Gardenia extract, in particular, has been reported to lead to cell apoptosis so that it inhibits aggressive tumor growth (Peng et al., 2005). However, there is no evidence concerning the effects of Gardenia fruit on the apoptotic pathway in oral cancer, or analyses regarding which fractions of Gardenia extracts are most effective in inducing cell death in oral cancer cells.

The purpose of the present study is to investigate the presence of DNA topoisomerase 1 inhibitor in various solvent fractions of Gardenia extract, and determine which fractions contribute most significantly to the apoptotic pathway and cytotoxic effects in oral cancer cells.

Materials and methods

Cell culture

Human oral cancer KB cell line and normal oral epidermal keratinocyte HaCaT cell line were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell lines were maintained at 1 × 10⁶ cells/mL in Dulbecco’s modified Eagle’s medium (DMEM, WelGENE, Daegu, Korea) supplemented with 10% heat inactivated fetal bovine serum, and incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Plant material extraction and fractionation

Dried Gardenia fruit was obtained from ERAPharm, Korea. The dried fruit (300 g) was ground into powder and extracted with 70% methanol at room temperature. The methanol extract was evaporated in a rotary vacuum evaporator (EYELA, Kubota, Japan). The extract (90 g) was suspended in water. The suspended extract was partitioned with n-hexane (Duksan, Ansan, Korea), dichloromethane (Merck, Darmstadt, Germany), ethyloxylacetate (Yakuri, Osaka, Japan), n-butanol (Yakuri) and aqueous fractions. Fractional extraction by n-hexane, dichloromethane, ethyloxylacetate and n-butanol yielded 1.085, 0.706, 1.427 and 23 g of the original amount of Gardenia, respectively. Finally, 50 g of the original amount of Gardenia remained in aqueous form. Before performing the bioassay, each fractional extract was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Loius, MO, USA) and diluted in DMEM.

DNA relaxation assay

To assay the inhibitory effect for DNA topoisomerase 1 of each Gardenia extract fraction, a DNA relaxation assay was performed by using a DNA topoisomerase 1 assay kit (TopoGEN, Port Orange, FL), according to the manufacturer’s instructions. The reaction mixture was prepared by adding pRYG DNA, topoisomerase 1 enzyme (Takara, Shiga, Japan), and individual fractions of Gardenia extract. After the incubation period, each mixture was loaded onto a 1% agarose gel, and electrophoresis was carried out.

Cell cytotoxic assay

Growing cells were plated in a 96-well plate at 1 × 10⁴ cells per well, and a serial dilution of individual Gardenia extract fractions was added to each well. Cytotoxicity was determined by performing the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye reduction assay as described by Mosmann (1983), with minor modifications.

Histomorphologic evaluation by Diff-Quick® staining

To analyze changes in cell morphology, KB cells were seeded in 6-well plates on cover slips at 1 × 10⁶ cells per well with 2 mL of medium in each well. After 24 h, the cover slips were allowed to air-dry for 10 min. The cover slips were then treated with Diff-Quick^® solution (International Reagent Co., Kobe, Japan) and mounted on a slide for visualization by light microscopy.

Flow cytometric assay

Cells were trypsinized as a single cell suspension, harvested by centrifugation, and washed with PBS. After fixation in ice-cold 70% ethanol at 4°C overnight, cells were collected and washed twice with PBS. The cells were then stained with propidium iodide (PI, 3 μg/mL) for 15 min. Apoptotic cells that exhibited fragmented DNA were then quantified using 1 × 10⁴ cells on a flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Caspase-3, caspase-8 and caspase-9 activity assay

Caspase activity was determined using a colorimetric caspase-3, caspase-8 and caspase-9 activities assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The protein concentration was determined using a BCA Protein Assay kit (Pierce Biotechnology, Rock-Ford, IL, USA). Caspase activities were measured by optical density at 405 nm, using a microplate reader (Bio-Rad, Hercules, CA, USA).

Western blot analysis

Whole cell lysates were separated in 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Amersham, Minneapolis, NJ, USA). The membrane was blocked with blocking solution [5% skim milk in TBST (2.42 g/L Tris-HCl, 8 g/L NaCl, 0.1% Tween 20 pH 7.6)] for 30 min. The membrane was incubated overnight at 4°C with rabbit polyclonal poly (ADP ribose) polymerase (PARP) antibody (Santa Cruz, CA) (1:2000) and goat polyclonal actin antibody (Santa Cruz) (1:1000). After a rinse with TBST, the membrane was incubated for 1 h with anti-rabbit and anti-goat horse radish peroxidase-conjugated (1:2000) secondary antibody. Finally, the immunoreactivity of the membrane bound proteins was detected using an enhanced chemiluminescence (ECL) detection kit (Amersham).

Statistical analysis

All experiments were carried out in triplicate. The results were subjected to analysis of variance (ANOVA) using SPSS for Windows version 12.0 (SPSS Inc., Chicago, IL, USA) for the analysis of differences.

Results

DNA topoisomerase 1 inhibition and cell cytotoxic assay of Gardenia extract

DNA topoisomerase 1 inhibitory activity of each Gardenia extract fraction was determined (Figure 1A). Each fraction (100 mg/mL) was assayed to evaluate the effect on topoisomerase 1 enzyme. The n-hexane and dichloromethane fractions inhibited DNA topoisomerase 1 effectively, thereby promoting the formation of supercoiled DNA.

Figure 1.  Inhibitory activity of Gardenia jasminoids Ellis extract on human DNA topoisomerase I (A). Lane a, 0.1 μg Supercoiled DNA without added enzyme; lane b, supercoiled DNA with 1 U DNA topoisomerase 1; lanes c-h, supercoiled DNA with 1 U DNA topoisomeraise 1 in the presence of (lane c) n-hexane, (lane d) dichloromethane, (lane 3) ethyloxylacetate, (lane f) n-butanol, (lane g) water fractions, and (lane h) methanol extract of gardenia. Cytoxic effects (B) of gardenia fractionated methanol extract on KB cells. MeOH, methanol; HX, hexane; DCM, dichloromethane; EtOAc, ethyloxylacetate; BuOH, butanol; and H₂O, water fractions. A comparison of the cytoxic effects of gardenia methanol extract (C) dichloromethane fraction, (D) on normal oral keratinocyte HaCat cells and oral cancer KB cells. The vertical bars indicate the means ± SD (n = 3). Significant difference at *P < 0.05 was compared to control.

The cytotoxic effects on KB cells were evaluated for each fraction over a range of concentrations: 0.2, 0.4, 0.6, 0.8, and 1 mg/mL (Figure 1B). No significant cytotoxicity against KB cells was observed at 0.2 mg/mL for ethyl oxyl acetate, n-butanol, or water fractions. However, 0.2 mg/mL methanol extract showed 40% growth inhibition. It was further observed that the dichloromethane fraction was the most effective at a concentration of 0.2 mg/mL against KB cells, showing growth inhibition of 91%.

The concentration-dependent cytotoxic effect of the methanol extract and dichloromethane fraction on HaCaT cells and KB cells was determined (Figure 1C and 1D). At a concentration of 0.1 mg/mL, the methanol extract showed no significant cytotoxicity against HaCaT cells or KB cells. However, 0.1 mg/mL dichloromethane fraction showed 77% growth inhibition against KB cells. Therefore, a concentration of 0.1 mg/mL dichloromethane fraction was selected for subsequent experiments, because cellular viability was easily assessed at this concentration.

KB cell death induced by dichloromethane fraction of Gardenia extract

The effect on KB cells of the dichloromethane fraction was measured over time (Figure 2A). Cells treated with Gardenia extract dichloromethane fraction (0.1 mg/mL) for 0, 3, 6, 12 and 24 h showed a time-dependent decrease in cellular viability. The cellular viability decreased to 65% and 10% after treatment of KB cells with dichloromethane fraction for 12 and 24 h, respectively.

Figure 2.  Cytoxic effects of gardenia dichloromethane fraction on KB cells. Cells viability, (A) histomorphologic evaluation, (B) and flow cytometric analysis (C) in KB cells treated with gardenia dichloromethane fraction. Cells were treated with 0.1 mg/ml dichloromethane extract for the indicated times and analyzed. All magnifications are × 200.

We further examined morphological features of KB cells treated with dichloromethane fraction to determine whether the fraction induced morphological changes known to be associated with apoptosis (Figure 2B). KB cells were treated with dichloromethane fraction (0.1 mg/mL) for 0, 3, 6, 12 and 24 h, and those cells treated for 24 h showed a reduced number of cells when compared to untreated control cells. Untreated cells displayed intact cellular morphology, but dichloromethane fraction-treated cells showed evidence of apoptosis, i.e., apoptotic bodies with membrane blebs characteristic of cells dying via apoptosis.

Apoptotic cell death is one mechanism by which cell growth is suppressed. Therefore, KB cells were treated with 0.1 mg/mL dichloromethane fraction for 0, 3, 6, 12, or 24 h, and apoptotic effects were monitored by flow cytometric analysis (Figure 2C). The Gardenia extract dichloromethane fraction clearly induced apoptosis in a time-dependent manner in KB cells; the relative percentage of apoptotic cells observed was 30.23%∼40.49%.

Activities of caspase-3, caspase-8, caspase-9 and PARP expression

The activity of caspase-3, caspase-8 and caspase-9 in KB cells treated with Gardenia extract dichloromethane fraction (0.1 mg/mL) was determined after 0, 3, 6, 12, and 24 h (Figure 3A). Caspase activities were increased by dichloromethane fraction incubation in a time-dependent manner. In particular, the activity of caspase-3 at 24 h was 500% higher than that of the control. It was further observed that the activity of caspase-8 and caspase-9 increased to 150% and 230%, respectively.

Figure 3.  Effect of dichloromethane fraction on capase activities and PARP protein expression. Caspase-3, caspase-8, and caspase-9 activity (A) and Poly (ADP ribose) polymerase (PARP) protein expression (B) by treatment of dichloromethane fraction on KB cells. Actin was used to ensure equal protein loading.

Cleavage of poly (ADP ribose) polymerase (PARP) is a typical feature observed in apoptotic cells. To understand the molecular mechanisms by which the dichloromethane fraction induced apoptosis, we examined PARP protein levels by western blotting (Figure 3B). When KB cells were treated with 0.1 mg/mL, a time-dependent increase in the formation of the 85 kDa fragment and decrease in the formation of the 116 kDa PARP was observed.

Discussion

DNA topoisomerase 1 is a ubiquitous and essential enzyme that relaxes DNA supercoiling inside cells during the progression of several vital cellular processes, such as replication, recombination, and transcription (Ganguly et al., 2007). Therefore, DNA topoisomerase 1 inhibitors and their associated pathways represent some of the most attractive targets for the development of anticancer therapeutics. Several drug discovery programs have produced potent, small molecules based on the plant toxin camptothecin that attack type I topoisomerases and bind to the complex of DNA and topoisomerase after enzymatic incision. This binding of the DNA/topoisomerase complex blocks the enzymatic reaction that normally reseals the DNA following its incision (Goodsell, 2002).

Gardenia jasminoides is one of the most widely used ingredients in traditional Oriental medicine for the treatment of febrile diseases, jaundice, acute conjunctivitis, epistaxis, hematemesis, pyogenic infections and ulcers of the skin, as well as sprains and painful swellings due to blood stasis (Lee et al., 2006). Biological and pharmacological activities such as the anti-tumor activity of Gardenia extract are especially remarkable, but neither the active component nor its molecular target(s) are well defined. Elucidation of the active component and its mechanism of action is likely to make such medicine more acceptable for use in a larger, more generalized population (Sandur et al., 2007).

In the present study, DNA topoisomerase 1 inhibition activity in the n-hexane and dichloromethane fractions of Gardenia extract was demonstrated. From this we infer that treatment with these fractions consequently promotes the formation of supercoiled DNA. Cytotoxic effects were observed in oral cancer KB cells after treatment with n-hexane and dichloromethane fractions, and the dichloromethane fraction showed the greatest cytotoxicity. KB cells were treated with 0.1 mg/mL dichloromethane fraction for various periods of time, and after 24 h almost all of the KB cells were dead. Many authors report geniposide and crocin as the primary active components of Gardenia, exhibiting cytotoxic and antitumor effects (Goodsell, 2002; Machida et al., 2000; Mathews-Roth, 1982; Ozaki et al., 2002; Tseng et al., 1995). Lee et al. (2005) reported that crocin was easily transformed to crocetin by the intestinal bacteria of humans and mice; in these experiments, geniposide and crocin were isolated from Gardenia by water extraction. However, in the present study, the cytotoxic effect on KB cells was observed in Gardenia extracts prepared from the dichloromethane partitioned fraction of a methanol extract. This demonstrates that components other than geniposide and crocin exhibited DNA topoisomerase 1 activity inhibition and resulted in the induction of cancer cell death. When the cytotoxic effects of the dichloromethane fraction in KB cells were compared with those in epidermal keratinocyte HaCaT cells, cytotoxic effects were observed only in KB cells. From a technical viewpoint, cancer chemotherapeutics are metabolic, microtubular, or chromosomal poisons that kill cancer cells by interfering with their ability to divide, and thus block tissue growth (Nguyen & Hussain, 2007). The therapeutic index of cancer chemotherapy depends on the selectivity of individual agents in targeting malignant cells versus normally dividing cells (Nguyen & Hussain, 2007). To assess this aspect in Gardenia extract, experiments comparing cytotoxicity of the dichloromethane fraction in KB cancer cells and HaCaT cells were performed. We found that “cell selectivity” is influenced by the fact that malignant cells are more mitotically active than normal cells, and thus can be poisoned more easily.

The dichloromethane fraction of Gardenia extract-induced cell apoptosis, which is morphologically characterized by chromatin condensation, DNA fragmentation, cytoplasmic membrane bleb, and cell shrinkage (Hockenbery et al., 1993; Ratan et al., 1994). To evaluate morphologic changes, KB cells were observed in a monolayer; as dichloromethane incubation time increased, so did the number of detached cells, indicative of cell death. After 24 h, apoptotic morphological features were identified. In flow cytometric analysis, the sub-G1 cell population was increased in a time-dependent manner, which indicated an increase in the number of apoptotic cells.

The apoptotic pathway is an active cell suicide mechanism consisting of an evolutionarily conserved cascade that converges on a family of cysteine-aspartases named caspases. To date, two major caspase pathways directed by caspase-8 or caspase-9 have been described and shown to mediate distinct sets of signals (Juang et al., 2004). Several caspases have been shown to be key executioners of apoptosis, mediated by various inducers including antitumor agents (Kaufmann, 1998). Recently, data have suggested that caspase-3 is involved in DNA topoisomerase 1, and microtubule inhibitor-induced apoptosis in tumor cells (Shimizu & Pommier, 1997; Tahir et al., 2001). In KB cells treated with Gardenia extract dichloromethane fraction, caspase-3 activity was shown to increase five-fold compared to that of untreated control cells, and this caspase-3 activation was accompanied by cleavage of PARP (116 kDa) into an 85-kDa fragment (Figure 3). These results demonstrated that treatment with Gardenia extract dichloromethane fraction induced apoptosis in KB cells, which is consistent with the findings of previous morphologic studies.

Conclusion

Gardenia extract exhibits DNA topoisomerase 1 inhibition activity, which presumably promotes the formation of supercoiled DNA. The cytotoxic effect of Gardenia extract dichloromethane fraction on KB oral cancer cells increased in a dose-dependent manner. Furthermore, this cytotoxicity was observed in the oral cancer KB cell line but not in the normal human epidermal keratinocyte HaCaT cell line. Treatment with the dichloromethane fraction of Gardenia extract induced apoptosis through an increase in caspase-3, caspase-8 and caspase-9 activities, and cleavage of poly (ADP-ribose) polymerase. These results present the possibility of Gardenia extract as a candidate for development as an anticancer therapy.

Declaration of interest

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2009-0069839).