Introduction
Cells undergoing apoptosis usually exhibit characteristic changes, including nuclear condensation and degradation of DNA into oligonucleosomal fragments [1]. Apoptotic cell death is thought to result ultimately from the proteolytic actions of caspase [2], and alterations in mitochondrial function play a key role in the regulation of apoptosis [3]. Moreover, the proteasome system has been implicated as a negative or positive mediator of apoptosis. The proteasome pathway is known to work mostly upstream of mitochondrial alterations and caspase activation [4].
Carcinoma of the oral cavity, especially oral squamous cell carcinoma (OSCC), is one of the leading causes of cancer-related death, affecting nearly 500,000 patients annually worldwide. OSCC is a major malignancy, which remains incurable with current therapies [5]. OSCC patients are treated with classical modalities consisting of surgery, radiotherapy, and/or chemotherapy. Given the high mortality rates with OSCC, new therapeutic approaches continue to be investigated. The most promising approach is the use of natural agents with known anticancer effects.
Curcumin is a natural component of the spice turmeric. It is derived from the rhizome of the Indian plant Curcuma longa and traditionally has been used in the treatment of digestion problems, parasites, gallstones, and arthritis in Southeast Asia [6]. Several studies have reported that curcumin has anti-oxidant, anti-inflammatory and anti-cancer effects in humans [7-9]. Other studies reported that curcumin inhibits cell proliferation and induces apoptosis in many cancer cells [10-12]. Cisplatin, which belongs to a class of platinum containing anti-cancer compounds, is an extremely potent anticancer agent, showing significant clinical activity against a variety of solid tumors [13]. It is a representative anticancer drug used to treat certain types of head and neck cancer, cervical carcinoma, lung cancer, neurological cancers, and a wide variety of other cancers. However, resistance to cisplatin remains a significant barrier to the survival of cancer patients.
Recent studies have demonstrated that co-treatment of an antitumor agent with a natural product with anticancer effects may be a potential therapeutic strategy to reduce the extent and the severity of cancer treatment-related toxicity [14-18]. A systemic study of synergistic apoptotic effects of co-treatment with curcumin and cisplatin on a human oral squamous carcinoma cell line is lacking. Therefore, the present study investigated the synergistic apoptotic effect of co-treatment with curcumin and the anticancer drug cisplatin on a human oral squamous cell carcinoma (SCC25) cell line.
Materials and methods
Reagents
The following reagents were obtained commercially: curcumin, cisplatin, dimethyl sulfoxide (DMSO), Hoechst 33342, RNase A, proteinase K, aprotinin, leupeptin, PMSF, thiazolyl blue tetrazolium bromide and propidium iodide (PI) were from Sigma (St. Louis, MO, USA); Mouse monoclonal anti-human caspase-3, caspase-7, poly(ADP-ribose) polymerase (PARP), cytochrome c, apoptosis-inducing factor (AIF) antibodies, and rabbit polyclonal anti-human DFF40 (CAD), DFF45 (ICAD), GAPDH antibodies, and FITC-conjugated goat anti-mouse and anti-rabbit IgGs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); HRP-conjugated sheep anti-mouse and anti-rabbit IgGs were from Amersham GE Healthcare (Little Chalfront, UK). 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide (JC-1) was from Molecular Probes (Eugene, OR, USA). Dulbecco's modified Eagle's medium (DMEM) and FBS were from Gibco (Gaithersburg, MD, USA). SuperSignal West Femto enhanced chemiluminescence Western blotting detection reagent was from Pierce (Rockford, IL, USA).
Cell culture
The SCC25 human oral saquamous cell carcinoma cell line was purchased from ATCC (Rockville, USA). Cells were maintained at 37°C with 5% CO2 in air atmosphere in DMEM/F12 with 4 mM L-glutamine, 1.5 g/l sodiumbicarbonate, 4.5 g/l glucose and 1.0 mM, sodiumpyruvate supplemented with 10% FBS.
Treatment of curcumin and cisplatin
Stock solutions of the curcumin (100 mM) and cisplatin (25 mg/ml) made by dissolving them in DMSO were kept frozen at -20°C until use. Twenty four hours after SCC25 cells were subcultured, the original medium was removed. The cells were washed with PBS and then incubated in the same fresh medium. Since single treatment of 5 μM curcumin and 4 μg/ml cisplatin for 24 h showed slight induction of cell death in MTT assay, this single concentration was utilized for further assessment of apoptosis for co-treatment.
MTT assay
SCC25 cells were placed in a 96-well plate and were incubated for 24 h. Then, the cells were treated with each drugs for various time points. After cells were treated with 500 μg/ml of thiazolyl blue tetrazolium bromide (MTT solution), they were incubated at 37°C with 5% CO2 for 4 h. And then the medium was aspirated and formed formazan crystals were dissolved in DMSO. Cell viability was measured by an ELISA reader (Tecan, Männedorf, Switzerland) at 570 nm excitatory emission wavelength.
Hoechst staining
Cells were harvested and cell suspension was centrifuged onto a clean, fat-free glass slide with a cytocentrifuge. The samples were stained in 4 μg/ml Hoechst 33342 for 30 min at 37°C and fixed for 10 min with 4% paraformaldehyde.
DNA electrophoresis
2 x 106 cells were resuspended in 1.5 mL of lysis buffer [10 mM Tris (pH 7.5), 10 mM EDTA (pH 8.0), 10 mM NaCl and 0.5% SDS] into which proteinase K (200 μg/ml) was added. After samples were incubated overnight at 48°C, 200 μl of ice cold 5 M NaCl was added and the supernatant containing fragmented DNA was collected after centrifugation. The DNA was then precipitated overnight at -20°C in 50% isopropanol and RNase A-treated for 1 h at 3 7°C. The DNA from 106 cells (15 μl) was equally loaded on each lane of 2% agarose gels in Tris-acetic acid/EDTA buffer containing 0.5 μg/ml ethidium bromide at 50 mA for 1.5 h.
Western blot analysis
Cells (2 x 106) were washed twice in ice-cold PBS, resuspended in 200 μl ice-cold solubilizing buffer [300 mM NaCl, 50 mM Tris-Cl (pH 7.6), 0.5% Triton X-100, 2 mM PMSF, 2 μg/ml aprotinin and 2 μg/ml leupeptin] and incubated at 4°C for 30 min. The lysates were centrifuged at 14,000 rpm for 15 min at 4°C. Protein concentrations of cell lysates were determined with Bradford protein assay (Bio-Rad, Richmond, CA, USA) and 50 μg of proteins were loaded onto 7.5-15% SDS/PAGE. The gels were transferred to nitrocellulose membrane (Amersham GE Healthcare, Little Chalfont, UK) and reacted with each antibody. Immunostaining with antibodies was performed using SuperSignal West Femto enhanced chemiluminescence substrate and detected with Alpha Imager HP (Alpha Innotech, Santa Clara, USA).
Immunofluorescence staining
Cells were cytocentrifuged and fixed for 10 min in 4% paraformaldehyde, incubated with each primary antibody for 1 h, washed 3 times for 5 min, and then incubated with FITC-conjugated secondary antibody for 1 h at room temperature. Cells were mounted with PBS. Fluorescent images were observed and analyzed under Zeiss LSM 700 laser-scanning confocal microscope (Göettingen, GER).
Assay of mitochondrial membrane potential (MMP)
JC-1 was added directly to the cell culture medium (1 μM final concentration) and incubated for 15 min. The medium was then replaced with PBS. Flow cytometry to measure MMP was performed on a CYTOMICS FC500 flow cytometry (Beckman Coulter, Brea, CA, USA). Data were acquired and analyzed using CXP software version 2.2.
Quantification of DNA hypoploidy by flow cytometry
After treatment for 24 h, cells were harvested by trypsinization and ice cold 95% ethanol with 0.5% Tween 20 was added to the cell suspensions to a final concentration of 70% ethanol. Fixed cells were pelleted, and washed in 1% BSA-PBS solution. Cells were resuspended in 1 ml PBS containing 20 μg/ml RNase A, incubated at 4°C for 30 min, washed once with BSA-PBS, and resuspended in PI solution (10 μg/ml). After cells were incubated at 4°C for 5 min in the dark, DNA content were measured on a CYTOMICS FC500 flow cytometry system (Beckman Coulter, FL, CA, USA) and data was analyzed using the Multicycle software which allowed a simultaneous estimation of cell-cycle parameters and apoptosis.
Results
Co-treatment with curcumin and cisplatin augmented the reduction in viability of SCC25 cells
To investigate whether co-treatment with curcumin and cisplatin reduced the viability of the SCC25 cells, an MTT assay was conducted. Single treatment of curcumin (5 μM) or cisplatin (4 μg/ml) for 24 h slightly reduced the viability of the SCC25 cells (curcumin, 94.6%; cisplatin, 93.4%). Co-treatment with curcumin and cisplatin significantly reduced the cell viability (45.2%) compared to the single treatment (Fig. 1). The concentration of DMSO used in this study had no effect on SCC25 cell proliferation in our preliminary studies.
Co-treatment with curcumin and cisplatin augmented the nuclear condensation and fragmantation in SCC25 cells
To explore whether nuclear condensation and fragmentation were induced by the co-treatment, Hoechst staining, a hallmark of apoptosis, was conducted. Hoechst staining showed slight nuclear condensation in response to the single treatment of curcumin and cisplatin. However, co-treatment with curcumin and cisplatin resulted in a variety of condensed and fragmented nuclei (Fig. 2A and 2B). To assess DNA fragmentation induced by the co-treatment, DNA electrophoresis was conducted. DNA electrophoresis did not show a ladder pattern of DNA fragments in the cells exposed to the single treatment of curcumin and cisplatin, whereas those exposed to the co-treatment exhibited a ladder pattern of DNA fragments (Fig. 2C).
Augmentation of apoptosis by co-treatment with curcumin and cisplatin in SCC25 cells
The flow cytometry results showed that the co-treatment with curcumin and cisplatin significantly increased the number of apoptotic cells with DNA hypoploidy compared to the single treatment (Fig. 3A). The co-treatment with curcumin and cisplatin resulted in the induction of the cleavage of caspase-3, caspase-7, and PARP and the production of DFF45/ICAD 11 kDa, PARP 85 kDa, and caspase-3 17 kDa cleaved products (Fig. 3B and Fig. 4A). The results of the confocal microscopy showed that DFF40/CAD was located in the cytosol following the single treatment with either curcumin or cisplatin, whereas it was clearly translocated into the nuclei following the co-treatment (Fig. 4B).
Augmentation of apoptosis by the co-treatment with curcumin and cisplatin was demonstrated by reduction of Bcl-2 and mitochondrial membrane potential (MMP) in SCC25 cells
The expression level of the antiapoptotic protein Bcl-2 decreased and the expression level of the apoptotic protein Bax increased in response to the co-treatment (Fig. 5A). The single treatment with either curcumin or cisplatin slightly reduced the MMP compared to the control group. In contrast, the co-treatment with curcumin and cisplatin resulted in a remarkable loss of MMP (Fig. 5B).
Co-treatment with curcumin and cisplatin caused the translocation of AIF from mitochondria into the nucleus
The results of confocal microscopy showed that AIF was located in the mitochondria in the cells that received the single treatment of curcumin and cisplatin, whereas it was evidently translocated into the nuclei in those that received the co-treatment (Fig. 6).
Co-treatment with curcumin and cisplatin caused the release of cytochrome c from mitochondria into the cytosol
The confocal microscopy results showed that cytochrome c was located in the mitochondria following the single treatment with either curcumin or cisplatin, whereas it was evidently released into the cytosol following the co-treatment (Fig. 7).
Discussion
Recently, the pharmacological mechanisms underlying the activities of many herbal remedies have been elucidated, and herbal medicine is becoming increasingly popular among health care professionals and the public. Curcumin is a widely used flavoring agent in food, and it has been reported to inhibit cell growth and induce apoptosis and to have antitumor activity in many cancers [19-21].
Since the 1970s, the popularity of the neutral, square planar, coordination complex cis-diamminedichloroplatinum (II) (cisplatin) in the treatment of cancer has increased. Cisplatin, which is a well-known DNA-damaging agent and one of the most active cancer treatment agents available, is widely used for the treatment of many malignancies, including testicular, ovarian, bladder, cervical, head and neck, and small-cell and nonsmall-cell lung cancers. Unfortunately, cisplatin produces many unwanted side effects [22,23]. Studies have reported that cisplatin-induced cell death is involved in apoptotic- and cell cycle- regulating pathways [24-28]. Moreover, the synergistic antitumor effect of combination treatment with cisplatin and antitumor agents or natural products has been demonstrated [29-32].
The mitochondria plays an important role in the induction of the mitochondrial permeability transition and also plays a key part in the regulation of apoptosis [3,33,34]. The outer mitochondrial membrane becomes permeable to intermembrane space proteins, such as cytochrome c and apoptosis-inducing factor (AIF) during apoptosis [35]. The release of cytochrome c and the disruption of the MMP are known features of apoptosis triggered by proteasome inhibition [36-38]. On induction of apoptosis, AIF translocates to the nucleus, resulting in chromatin condensation and large-scale DNA fragmentation [39,40]. This study clearly showed that co-treatment of the SCC25 cells with curcumin and cisplatin results in a remarkable decrease in the MMP, an increase in Bax, and a decrease in Bcl-2, as well as the release of cytochrome c into the cytosol and the translocation of AIF to the nuclei. In contrast, these patterns were not observed in the SCC25 cells in response to the single treatment.
A common final event in apoptosis is nuclear condensation, which is controlled by caspases, DFF, and PARP. Caspases, cysteinyl aspartate-specific intracellular proteinases, play an essential role during apoptotic death [41]. Once activated, the effector caspases (caspase-3, caspase-6, or caspase-7) result in proteolytic cleavage of a broad spectrum of cellular targets, ultimately leading to cell death. The known cellular substrates include structural components (such as actin and nuclear lamin), inhibitors of deoxyribonuclease (such as DFF45/ICAD), and DNA repair proteins (such as PARP) [42-44]. In apoptotic cells, the activation of DFF40/CAD, also a substrate of caspase-3, occurs with the cleavage of DFF45/ICAD. Once DFF40/CAD is activated and released from the complex of DFF45/ICAD and DFF40/CAD, it can translocate to the nucleus and then degrade chromosomal DNA and produce DNA fragmentation. This study demonstrated that co-treatment of SCC25 cells with curcumin and cisplatin results in the degradation and the cleavage of caspase-3, caspase-7, PARP, and DFF45/ICAD, in addition to the translocation of DFF40/CAD to nuclei, whereas the single treatment does not.
Taken collectively, the combination therapy with curcumin and cisplatin could be considered in the future as an alternative therapeutic strategy for human oral squamous carcinoma. Further extensive studies are needed prior to its clinical application.
