Introduction
Salivary gland tumors are a morphologically and clinically diverse group of neoplasms, which may present considerable diagnostic and management challenges to the pathologist or surgeon [1]. Salivary gland tumors are rare with an overall incidence in the Western world of about 2.5 - 3.0 per 100,000 per year [2]. Among salivary gland tumors, the salivary gland epidermoid cell carcinomas are more rare [2]. However, although this tumor has an extremely poor prognosis, unlike most other salivary gland malignancies, survival at 5 years is prognostically significant [2]. Therefore, an understanding of the molecular mechanisms of salivary gland epidermal carcinoma is one of the most important issues for treatment. New therapeutic strategies are necessary to increase survival rates in patients with salivary gland epidermal carcinomas.
Recent studies have shown that such chemicals derived from natural materials have been identified to elicit chemopreventive and therapeutic effects [3-5]. It has been reported that this effect alters various factors associated with the cell cycle and thereby induce the apoptotic cell death [3-7]. Therefore, induction of apoptosis in cancer cells has become an important indicator of the cancer treatment response in employing a bioactive substance to reduce and control human mortality due to cancer [8,9]. There is a need to find new anti-cancer agents that can kill cancerous cells with minimal toxicity.
Apoptosis, which is a major way of programmed cell death, plays an important role in the regulation of tissue development and homeostasis in eukaryotes [10-12]. Apoptosis may occur via a death receptor-dependent extrinsic or a mitochondriadependent intrinsic pathway and apoptosis is induced by treatment of chemotherapeutic agents [13,14].
Curcumin (diferuloylmethane), a constituent of turmeric powder derived from the rhizome of Curcuma longa, has antiinflammatory, antimicrobial, antioxidative, immunomodulating and antiatherogenic properties [15-18]. Many studies provide the fact that curcumin has chemopreventive and antiproliferative activity in various types of human cancers [18-23]. Furthermore, curcumin is also pharmacologically safe as it is a naturally occurring compound [24,25]. Unfortunately, natural curcumin has been limited the use because of its poor intestinal absorption [26]. Therefore it remains an excellent compound for the design of more effective analogs. One monoketone analog, EF-24 (diphenyl difluoroketone), is efficacious in anticancer screens [27,28]. Although it is shown to reduce cancer cell viability, its action mechanisms remain to be elucidated. In addition, the effects of EF-24 and curcumin on salivary gland epidermal carcinoma are not clearly established.
In this study, therefore, the effects of EF-24 and curcumin on cell growth and the mechanism of cell death elicited by EF-24 and curcumin were examined in HTB-41 human submaxillary salivary gland epidermoid carcinoma cells.
Materials and Methods
Drugs
EF-24 and curcumin (Fig. 1) were supplied by Sigma (St Louis, MO, USA). Anti-cleaved caspase-3, anti-cleaved caspase-7 and anti-cleaved caspase-9 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The cell-permeable fluorogenic substrate PhiPhiLux-G1D2 was purchased from OncoImmunin, Inc. (Gaithersburg, MD, USA). Other analytical reagents were purchased based on the analytical grade.
Cell line and cell cultures
HTB-41 human submaxillary salivary gland epidermoid carcinoma cells were provided by American Type Culture Collection (ATCC, Rockville, MD, USA). The HTB-41 cells were grown in modified McCoy's media with 1.5 mM L-glutamine adjusted to contain 2.2 g/L sodium bicarbonate with 10% FBS in accordance with ATCC's instruction. The cells were maintained as monolayers in plastic culture plate at 37℃ in a humidified atmosphere containing 5% CO2.
MTT assay
The cell viability test was performed according to the previously described method with minor modifications [29,30]. The HTB-41 cells were seeded at a concentration of 5 X 103 cells/well in a 24-well plate. After 24 hours growth, the cells were treated with EF-24 or curcumin at various concentrations and incubation times. Then, cell viability was assessed using MTT assay. Briefly, the cells were grown in the medium in the absence or presence of EF-24 or curcumin for various incubation times. Following the culture, 0.5 mg/ml of MTT was added to each well. After 4 hours incubation at 37℃, isopropanol with 0.04 M HCl was added to each well to dissolve precipitates. Then, the absorbance was measured at 570 nm using a spectrophotometer (Ultrospec 2000; Amersham Pharmacia Biotech, NJ, USA). Four or five separate experiments were performed for each concentration/ exposure time combination.
Nuclear staining with DAPI
Nuclear staining with DAPI (4’,6-diamidino-2-phenylindol) was performed to evaluate apoptosis. The HTB-41 cells were cultured in 24-well plates at a seeding density of 5 X 103 cells per well. After 24 hours growth, the cells were treated with 3 μM EF-24 or 100 μM curcumin for 24 hours. The treated HTB-41 cells were fixed with 1% paraformaldehyde for 30 min at room temperature and washed twice with PBS. Permeate the cells with ice-cold ethanol for 5 min at room temperature and washed twice with PBS. The fixed HTB-41 cells were stained with DAPI (300 nM) for 5 min at room temperature in dark, washed twice with PBS and examined by fluorescent inverted microscopy (IX71, Olympus, Japan).
Immunoblotting
The HTB-41 cells were treated with 3 μM EF-24 or 100 μM curcumin for 24 or 48 hours. Immunoblotting was performed according to the previously described method with minor modifications [31,32]. The anti-cleaved caspase-3, -7 or -9 antibodies (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) were used as the primary antibodies.
Determination of caspase activation
The activity of caspase-3/-7 was determined using the cell-permeable fluorogenic substrate PhiPhiLux-G1D2 (OncoImmunin, Inc. Gaithersburg, MD, USA), which was used according to the manufacturer’s instructions. The HTB-41 cells were treated with 3 μM EF-24 or 100 μM curcumin for 24 hours and incubated with PhiPhiLux-G1D2. The activity of caspase-3/-7 was visualized by fluorescence microscopy (IX71, Olympus, Japan).
Data analysis
All experiments were performed at least four times. The results were presented as the mean ± SEM. The statistical significance was analyzed by using Student's t-test for the two group comparison and one way analysis of variance for the multi-group comparisons. A p value <0.05 was considered statistically significant. All statistical analyses were performed using Excel and SPSS software.
Results
Growth inhibition of HTB-41 cells by EF-24 and curcumin
To analyze and compare the effect of EF-24 and curcumin on the viability of HTB-41 cells, the cells were treated with EF-24 and curcumin at various concentrations for 24 and 48 hours, and then the MTT assay was performed. As shown in Fig. 2, treatment of curcumin from 0.1 to 30 μM for 24 hours or from 0.1 to 3 μM for 48 hours did not significantly affect the cell viability of HTB-41 cells, but curcumin 100 μM for 24 hours or curcumin 10, 30 and 100 μM for 48 hours reduced HTB-41 cell viability. When the HTB-41 cells were treated with EF-24 from 0.1 to 10 μM for 24 or 48 hours, EF-24 inhibited the growth of HTB-41 cells in the concentration- and time-dependent manners, suggesting that curcumin and EF-24 induce HTB-41 cell death (Fig. 2). The IC50 values of curcumin and EF-24 on the cell viability after 24 or 48 hours treatments are shown in Table 1. The apparent potency of EF-24 was > 34 times that of curcumin. More importantly, the effects were observed at an EF-24 concentration < 3 μM, a concentration at which curcumin had no significant effect on cell proliferation, indicating the enhanced potency of EF-24.
Changes in nuclear morphology by EF-24 and curcumin
The nuclear morphological changes were assessed by DAPI staining. As shown in Fig. 3A, the nuclei of the control HTB-41 cells (Mock) had a normal regular and oval shape. Treatment with 3 μM EF-24 or 100 μM curcumin for 24 hours resulted in nuclear condensation and fragmentation, which are characteristics of apoptosis. As quantified in Fig. 3B, EF-24 and curcumin significantly increased the apoptotic rate of HTB-41 cells to 45.9 ± 3.1% and 31.8 ± 1.7%, respectively.
Activation of caspases by EF-24 and curcumin
The levels of cleaved caspase-3, -7 and -9 were examined by immunoblotting and the activity of caspase-3/-7 was detected by fluorescence microscopy using a selective fluorogenic substrate since caspase-3, -7 and -9 are effector caspases of apoptotic cell death [33-36]. Treatment with 3 μM EF-24 or 100 μM curcumin for 24 or 48 hours significantly promoted the proteolytic cleavages of procaspase-3 in the HTB-41 cells, with the increases in the amount of cleaved caspase-3 (Fig. 4). Either EF-24 (3 μM) or curcumin (100 μM) for 24 or 48 hours also promoted the proteolytic cleavages of procaspase-7, with the increases in the amount of cleaved caspase-7 (Fig. 5). Treatment with EF-24 (3 μM) or curcumin (100 μM) promoted the proteolytic cleavages of procaspase-9 in the HTB-41 cells (Fig. 6).
In addition, activation of caspase-3/-7 in EF-24 or curcumin treated HTB-41 cells was confirmed by fluorescence microscopy using a fluorogenic substrate. As shown in Fig. 7, either the EF-24 or curcumin treatment led to activate the caspase-3/-7 in the living HTB-41 cells.
Discussion
In recent years, there has been a global trend toward the importance of naturally occurring phytochemicals in plants for the prevention and treatment of human diseases [37,38]. Several of these phytochemicals are shown to have potential values as cancer chemopreventive or therapeutic agents within the human body [37-40]. Most of these bioactive phytochemicals exert their cancer chemotherapeutic activity by blocking cell cycle progression and triggering apoptotic cell death [37-40].
Curcumin has been extracted from the dried ground rhizome of the perennial herb Curcuma longa. Several studies have suggested that curcumin induces cell cycle arrest and apoptosis in various cancer cells [18-23]. However, it is needed the design of more effective analogs because of curcumin's poor intestinal absorption [26]. EF-24, the monoketone analog of curcumin (Fig. 1), is efficacious in anticancer screens and has been shown to inhibit the growth of human breast tumor xenografts in a mouse model [27,28]. Although EF-24 can reduce cancer cell viability, the mechanisms of action remain to be elucidated, and the effects of EF-24 and curcumin on salivary gland epidermoid cell carcinoma are not clearly established. In this study, therefore, the cytotoxic effect of EF-24 and the mechanism of cell death exhibited by EF-24 were examined in HTB-41 human submaxillary salivary gland epidermoid carcinoma cells. The results of this study indicate that EF24, a novel curcumin analog, possesses profound promise as an anti-salivary gland cancer therapeutic.
In MTT assay, curcumin 100 μM for 24 hours or curcumin 10, 30 and 100 μM for 48 hours reduced HTB-41 cell viability (Fig. 2). EF-24 inhibited the growth of HTB-41 cells in the concentration- and time-dependent manners (Fig. 2). This corresponded with the results of EF-24 and curcumin that have anti-cancer effects via the suppression of cancer cell growth in various types of cancer cells [18-23]. Also, the apparent potency of EF-24 was > 34-fold that of curcumin. These results speculate that EF-24 and curcumin have cytotoxicity to salivary gland epidermoid carcinoma cells with EF-24 having enhanced potency. In addition, these results indicate that it has potential value for anti-cancer drug discovery.
The induction of apoptosis in cancer cells is one of useful strategies for anti-cancer drug development [31]. So, many studies were performed for screening of apoptosis from plant-derived compounds [37-40]. In this study, treatment with EF-24 and curcumin induced nuclear condensation and fragmentation in HTB-41 cells, suggesting apoptotic cell death (Fig. 3). These results indicate that EF-24 and curcumin inhibit the growth of HTB-41 cells by activating cell apoptosis.
The activation of a family of intracellular cysteine proteases, called caspases, is known to play an important role in the initiation and execution of apoptosis induced by various stimuli [34,35]. Among the caspases identified in mammalian cells, caspase-3, caspase-7 and caspase-9 may serve as effector caspases of apoptotic cell death [33-35]. Caspase-3, caspase-7 and caspase-9 are synthesized as inactive proenzymes (of sizes 32 kDa, 35 kDa and 47 kDa, respectively), which require proteolytic activation to cleaved enzymes (of sizes 19 kDa, 20 kDa and 37 kDa, respectively) [33-35]. This study revealed that low levels of cleaved capase-3, -7 and -9 were present in EF-24- or curcumin-untreated HTB-41 cells, and the amount of cleaved enzymes was increased after the EF-24 or curcumin treatment in HTB-41 cells (Fig. 4, 5 and 6). In addition, the activity of caspase-3/-7 was increased by EF-24 or curcumin treatment in living HTB-41 cells compared to dimethyl sulfoxide (DMSO) treatment as a control (Fig. 7). These results suggested that EF-24 and curcumin induce apoptotic cell death through caspase-3-, -7- and -9-dependent processing in the HTB-41 cells. However, the mechanisms of apoptosis induced by EF-24 and curcumin in HTB-41 cells are not yet completely understood. Further studies will be needed to reveal the precise cellular and molecular mechanisms of apoptosis induced by EF-24 and curcumin.
In conclusion, these results indicate that the EF-24 and curcumin inhibit cell proliferation and induce apoptosis in HTB-41 human salivary gland epidermoid carcinoma cells. Moreover, the potency of EF-24 was > 34 times that of curcumin. Therefore, EF-24 could be a model compound for the further development of natural product-derived anti salivary gland epidermoid carcinoma agents. However, to elaborate this nascent possibility, further investigation of its activity including in vivo and purification of bioactive compounds is now in progress.
