Introduction

Shikonin is a natural naphthoquinone derivative compound made from the root of Lithospermum erythrorhizon, which has been used as a traditional herbal medicine in China to treat external wounds, burns, and dermatitis for thousands of years. It has also demonstrated diverse biological activities, including anti-inflammatory, antioxidant, antimicrobial, antifungal, and anticancer effects [1,2]. More recently, it has been reported that shikonin exerts an anticancer effect on various cancer cell lines [3-7]. Apoptosis, which is an essential mechanism of cell death induced by anticancer drugs, is required for normal embryonic development, tissue homeostasis, and suppression of tumorigenesis [8-10]. Typical events of apoptosis are cell shrinkage, plasma membrane blebbing, chromatin condensation, and fragmentation [11]. In general, apoptosis has been divided into two major pathways-extrinsic and intrinsic pathways. The intrinsic pathway of apoptosis is mediated by mitochondria [12].

Worldwide, oral squamous cell carcinoma (OSCC) is one of the most common cancers occurring in the oral cavity [13-15]. While OSCC patients are currently treated with the classical modalities, consisting of surgery, radiotherapy, and/or chemotherapy [16-18], this malignancy is difficult to cure using current therapies [19], and it continues to result in high mortality rates. Therefore, new therapeutic approaches have been investigated, and the use of natural agents is among the most promising anticancer treatments.

Although a few studies have examined the apoptosisinducing efficacy of shikonin on cancer cells in vitro, no reports have been published regarding the apoptotic effect of shikonin on a human tongue squamous cell carcinoma cell line. The present study was conducted to examine the cytotoxicity and cell growth inhibition, as well as to explore the molecular mechanisms underlying the altered expression of cell cycle-related proteins and the induction of apoptosis, in the SCC25 human tongue squamous carcinoma cell line treated in vitro with shikonin.

Materials and Methods

Cell culture and treatment of shikonin

The SCC25 human tongue squamous cell carcinoma cell line was purchased from ATCC (Rockville, MD) and incubated at 37°C with 5% CO₂ in air atmosphere in DMEM/F12 medium with 2.5 mM L-glutamine and 15 mM HEPES buffer supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). The cells were grown on culture dishes or several types of plates for 24 hours. Then, the original medium was removed and the cells were washed with phosphate-buffered saline (PBS). Fresh medium was added to the plates, after which 100 mM of shikonin stock (Calbiochem, La Jolla, CA) was added to the medium to obtain 0.1-10 μM concentrations of the drug.

MTT assay

The cells were plated in 96-well culture plates and left overnight, after which they were treated with various concentrations of shikonin for various periods of time. At the termination of treatment, the cells were treated with 500 μg/ml of MTT stock solution (Sigma, St. Louis, MO) and incubated at 37°C with 5% CO₂ for four hours. The formed formazan crystals were dissolved in 150 μl of dimethyl sulfoxide (Sigma).

Clonogenic (colony-forming) assay

The cells were seeded in a 6-well plate at a density of 2.5×10²/well and grown overnight. They were then treated with 0, 0.1, 0.25, 0.5, 1.0 and 2.5 μM of shikonin and allowed to grow for 1 week. The colonies were then fixed in 100% methanol and stained with 0.5% (w/v) crystal violet (Sigma) for 10 minutes, after which they were washed with tap water and dried at room temperature. The colonies were scored and imaged under an IMT-2 inverted microscope (Olympus, Tokyo, JPN). Clonogenic survival was expressed as the percentage of colonies formed in shikonin-treated cells compared to a control.

Hoechst staining

Vehicle- and shikonin-treated cells were harvested and cytocentrifuged onto a glass slide. The cells were stained with 10 μg/ml Hoechst 33342 (Sigma) for 10 minutes at 37°C in the dark and washed with PBS. The slides were mounted with glycerol and the cells were examined under a fluorescence microscope (Carl Zeiss, Göettingen, Germany). The number of cells that showed a condensed or fragmented nucleus was determined from a random sampling of 3×10² cells per experiment by a blinded observer.

DNA electrophoresis

Cell samples (2×10⁶) were collected and lysed with lysis buffer [10 mM Tris (pH 7.5), 10 mM EDTA (pH 8.0), 10 mM NaCl, and 0.5% SDS] into which 200 μg/ml proteinase K (Sigma) was added. After the samples were incubated overnight at 48°C, 200 μl of ice-cold 5 M NaCl were added, and the supernatant containing fragmented DNA was collected after centrifugation. The DNA was then precipitated overnight at -20°C in 50% isopropanol and treated with RNase A for 1 hour at 37°C. The samples were equally electrophoresed in each lane of 2% agarose gels and visualized by ethidium bromide staining.

Western blot analysis

The shikonin-treated cells were washed twice with ice-cold PBS and resuspended in solubilizing buffer [300 mM NaCl, 50 mM Tris-HCl (pH 7.6), 0.5% TritonX-100, 2 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml leupeptin (Sigma)] and incubated at 4°C for 1 h. The lysates were centrifuged at 14,000 rpm for 30 minutes at 4°C. Protein concentrations of the cell lysates were determined with Bradford protein assay (Bio-Rad, Richmond, CA). A sample of 50 μg of protein in each well was separated and loaded onto 7.5–15% SDS/PAGE. The gels were transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA) and reacted with each antibody. Human anti-AIF (Upstate Laboratories, Syracuse, NY), cytochrome c, caspase-9, -7, -6, -3, Bax, Bcl-2, Lamin A/C, DFF45 (ICAD), p27^KIP1, CyclinD1, CyclinD3, Cdk2, Cdk4, poly (ADP-ribose) polymerase (PARP), GAPDH (Sigma), and DFF40 (CAD) (Stressgen, Ann Arbor, MI) antibodies were used at a 1:1000 dilution as the primary antibodies. FITC-conjugated goat anti-mouse and anti-rabbit immunoglobulin G (IgG) (Sigma) and HRP-conjugated sheep anti-mouse and anti-rabbit IgG (Amersham, GE Healthcare, Little Chalfont, UK) were used as the secondary antibodies at a 1:5000 dilution. The protein bands were visualized with SuperSignal West Femto enhanced chemiluminescence substrate (Pierce, Rockford, IL) and an Alpha Imager HP (Alpha Innotech, Santa Clara, CA).

Measurement of mitochondrial membrane potential (MMP)

DiOC₆ (Molecular Probes, Eugene, OR) was added to the medium (1 μM final concentration) and incubated for 30 minutes. To investigate MMP, flow cytometry was performed using a CYTOMICS FC500 system (Beckman Coulter, Brea, CA). The data were analyzed using CXP software version 2.2. The analyzer’s threshold on the FSC channel was adjusted to exclude noise and most of the subcellular debris.

Immunofluorescent staining

The cells were plated on coverslips for 1 day and then used for stimulation or staining with MitoTracker red (50 nM). The cells were then washed twice with PBS, fixed with 4% Paraformaldehyde (PFA) in PBS for 15 minutes, and washed 3 times with PBS. After permeabilization with 0.1% Triton X-100 in PBS and blocking with 1% BSA (Sigma) in PBS for 30 minutes, the cells were treated with primary antibodies in 1% BSA overnight at 4°C.

Then, the cells were washed with PBS and incubated with FITC-conjugated secondary antibodies in 1% BSA-PBS for 1 h. The fluorescent images were analyzed under a Zeiss LSM 750 laser-scanning confocal microscope (Carl Zeiss).

Flow cytometry analysis

The cells were seeded in 60-mm dishes at 70% confluence and incubated overnight. The shikonin-treated cells were incubated for different periods. The harvested cells were washed with PBS containing 1% BSA and centrifuged at 2500 rpm for 10 minutes. The cells were then resuspended in ice-cold 95% ethanol with 0.5% Tween 20 to a final concentration of 75% ethanol. After 24 hours, the fixed cells were washed in 1% BSA-PBS solution, resuspended in 1 ml PBS containing 40 μg/ml RNase A (Sigma), incubated at 4°C for 30 minutes, and resuspended in 10 μg/ml PI solution (Sigma). The DNA contents were examined using a CYTOMICS FC500 flow cytometer (Beckman Coulter) and the data were analyzed using MultiCycle software, which allowed a simultaneous estimation of cell-cycle parameters and apoptosis.

Results

Effects of shikonin on the cell viability and proliferation of SCC25 cells

The cytotoxicity of shikonin was determined by measuring the viability of SCC25 cells using the MTT assay. After shikonin treatment (0–10 μM) for 24–72 hours, the viability of the SCC25 cells was reduced by 99.0% (1 μM) to 29.4% (10 μM) at 24 hours; the reduction also showed time dependence (Fig. 1). The half-maximal inhibitory concentration (IC₅₀) of shikonin was 5 μM at 48 hours; this concentration was accepted for further analysis of apoptosis and alteration of cell cycle-related proteins. A clonogenic assay was used to investigate whether shikonin inhibited the growth of SCC25 cells. After exposure to shikonin concentrations (0–2.5 μM) for seven days, the SCC25 cells were examined for inhibition of colony formation, as shown in Fig. 2. Colony growth in the shikonin-treated group was determined as a percentage of the control’s untreated cell growth. The values for colony formation were 101.8% (0.1 μM), 111.4% (0.25 μM), 68.5% (0.5 μM), 43.2% (1 μM), and 5.1% (2.5 μM).

Morphological and biochemical changes in shikonin treated SCC25 cells

Treatment with shikonin resulted in morphological and biochemical changes associated with apoptosis. Hoechst staining demonstrated that shikonin induced a change in nuclear morphology. The untreated SCC25 control cells had round nuclei, whereas the SCC25 cells treated with 2.5–10 μM of shikonin for 24 hours displayed condensed and fragmented nuclei (Fig. 3). DNA fragmentation (DNA ladders), which is the important hallmark of apoptosis, was demonstrated by DNA electrophoresis. Electrophoresis of DNA from the SCC25 cells treated with different concentrations of shikonin revealed DNA ladders (Fig. 4). The apoptotic percentages were evaluated by flow cytometry analysis; shikonin treatment significantly increased the numbers of apoptotic cells with DNA hypoploidy in a dose-dependent manner (Fig. 5).

Shikonin activates Mitochondrial apoptotic event and caspase cascade

The induction of apoptosis is controlled by members of the Bcl-2 protein family. Bcl-2 has an antiapoptosis function, whereas Bax promotes apoptosis. The members of the proapoptotic Bcl-2 family, such as Bax, Bid, and Bad also induce the collapse of mitochondrial membrane potential (△Ψ m) and promote the release of cytochrome c and AIF. The function of Bcl-2 family proteins in shikonin-induced apoptosis was examined by Western blot assays. Bax was upregulated and Bcl-2 was downregulated by shikonin in a dose-dependent manner (Fig. 6). The mitochondria were stained with DiOC₆, and the mitochondrial membrane potential (△Ψm) was measured by flow cytometry. The SCC25 cells treated with shikonin showed a loss of mitochondrial membrane potential (△Ψm) in a dose-dependent manner (Fig. 7). The release of AIF and cytochrome c from the mitochondria was examined by confocal microscopy, and it was found that AIF was translocated from the mitochondria to the nucleus and cytochrome c was released from the mitochondria into the cytoplasm in SCC25 cells treated with 5 μM shikonin (Figs. 8 and 9).

Western blot assays showed that shikonin treatment at different concentrations induced degradation of caspase-9, caspase-6, PARP, and lamin A/C, and that it promoted production of caspase-3 17 kDa, as well as DFF45 34 kDa and 11 kDa cleaved products (Figs. 10). Confocal microscopy showed that shikonin treatment promoted the translocation of DFF40 (CAD) from the cytoplasm into the nucleus (Fig. 11).

Shikonin induces cell cycle arrest at G1 phase

To investigate the distribuition of cell cycle and expression levels of cell cycle-related proteins, flow cytometry analysis and western blot assays were performed. The results showed that the population of apoptotic cells were increased and the expression levels of Cyclin D1, Cyclin D3, Cdk4, and Cdk2 regulating the G0/G1 phase decreased in a time-dependent manner. The cdk inhibitor p27^KIP1was markedly upregulated (Fig.12-13).

Discussion

Natural products from plants used in traditional Chinese medicine and Ayurvedic medicine are currently being used more extensively [20, 21]. The pharmacological benefits of herbal medicines are becoming well known, and a number of studies have demonstrated that individual herbal medicines have antioxidant, antiallergic, antipyretic, anti-inflammatory, and antitumor effects [22-28].

It has been reported that one such substance, shikonin, which is derived from the rhizomes of turmeric, inhibits the proliferation of various tumor cells [3-7, 29]. Also, shikonin has prevent chondrocytes from IL-1β-induced apoptosis suggesting that shikonin has a positive effect on human normal cells [30]. As such, the present study investigated the effects of shikonin on the viability of SCC25 cells. The results revealed that shikonin produced in a dose- and time-dependent reductions in viability as determined by MTT assay. In addition, a clonogenic assay confirmed that 0.1–2.5 μM of shikonin markedly inhibited the growth of SCC25 cells. These data suggest that shikonin exerts a specific cytotoxic effect on SCC25 cells.

Apoptosis and necrosis are ultimately distinguishable forms of cell death that can be discriminated by their specific morphological changes. During apoptosis, cells undergo morphological changes that include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [31, 32]. In this study, Hoechst staining and DNA electrophoresis of SCC25 cells treated with shikonin revealed apoptotic hallmarks such as the formation of apoptotic bodies and DNA ladders. These results indicate that shikonin induced SCC25 cell death by activating apoptosis.

The mitochondrial pathway plays a crucial role in apoptosis and can be stimulated by various cellular stress signals. When cells experience an apoptotic stimulus, the proapoptotic protein Bax is activated or the antiapoptotic protein Bcl-2 is inactivated [33-37]. This activation/inactivation of Bax/Bcl-2 proteins induces changes in the mitochondrial membrane that lead to the dissipation of the inner membrane potential and permeabilization of the outer membrane of the mitochondria. This process induces the release of various proapoptotic proteins, in turn, such as cytochrome c, Smac/Diablo, endonuclease G, and AIF. Our results showed a significant shift in the ratio of Bax to Bcl-2 and a loss of mitochondrial membrane potential in SCC25 cells treated with shikonin. These results indicate that the change in Bax to Bcl-2 ratio could be due to molecular mechanisms by shikonin-induced apoptosis of the SCC25 cells.

Many studies have noted that the proapoptotic Bcl-2 family induces the collapse of mitochondrial membrane potential and the release of cytochrome c and AIF [38]. Generally, cytochrome c is translocated into the cytoplasm and binds to Apaf-1 during apoptosis; the cytochrome c/Apaf-1 complex promotes the activation of caspase-9. Procaspase-3 is then affected by caspase-9 and a caspase cascade is initiated and activated [39]. The AIF released through activation of the proapoptotic Bcl-2 family is translocated to the nucleus, resulting in morphological changes in the nucleus, such as chromatin condensation and DNA fragmentation [40]. In the present study, shikonin treatment induced the translocation of AIF from the mitochondria into the nucleus, cytochrome c release from the mitochondria into the cytosol, the cleavage of caspase-9, and a significant loss of MMP. These data clearly demonstrate that shikonin-induced apoptosis in the SCC25 cells involved the hallmark mitochondrial events.

The initiator caspases (e.g., caspases-2, -8, and -9) are activated by distinct proapoptotic stimuli to induce the effector caspases (e.g., caspase-3, -6, or -7), which perform a proteolytic cascade function of a broad spectrum of cellular targets, ultimately resulting in cell death [41-44]. A common final event of apoptosis is nuclear condensation, which is controlled by caspases, DFF (DNA fragmentation factor), and PARP. In apoptotic cells, the activation of DFF40 (CAD), which is a substrate of caspase-3, produces the cleaved form of DFF45 (ICAD). Once degraded DFF40 is released from the DFF45/DFF40 complex and translocated to the nucleus, chromosomal DNA is degraded to produce DNA fragments [45]. In the present study, shikonin treatment of SCC25 cells led to the activation of caspase-3, which in turn led to the increased cleavage of caspase-6, -7, and -9, followed by changes in PARP, lamin A/C, and DFF45 (ICAD). Confocal microscopy showed that shikonin treatment promoted the translocation of DFF40 (CAD) from the cytoplasm to the nucleus. Therefore, these data demonstrated that shikonin-induced apoptosis in SCC25 cells is associated with caspase cascades and the inactivation of the DFF40 (CAD) / DFF45 (ICAD) complex, as well as the translocation of DFF40 (CAD) from the cytoplasm into the nucleus and degradation of the chromosomes into fragments.

Molecular analysis in previous studies has revealed mutations of cell cycle regulators in most common malignancies. Therefore, regaining the regulation of cell cycle distribution is a potential strategy for anticancer therapy. The Cdks, Cyclins, and Cdk inhibitors play critical roles in the distribution of the cell cycle; the active Cdk-Cyclin complex is affected by Cdk inhibitors [46]. For example, it has been demonstrated that p21^WAF1/CIP1 and p27^KIP1 play an important role by binding to Cdk4/Cyclin D and Cdk2/Cyclin E complexes and preventing activation of these complex in regulating transition at the G1/S phase [47, 48]. Cell cycle G1 arrest is known to be related to activation of the p53 tumor suppressor protein, which acts as a transcription factor and regulates the expression of several components implicated in the pathways that regulate cell cycle progression and apoptosis [49, 50]. In this study, Cdk2, Cdk4, Cyclin D1, and Cyclin D3 were markedly downregulated, whereas p27^KIP1 was markedly upregulated. These data demonstrate that shikonin-induced apoptosis in SCC25 cells involved alterations in the expression of G1 cell cycle-related proteins. Moreover, p27^KIP1 might play a key role in shikonin-induced SCC25 cell death.

Taken collectively, the results of this study demonstrate that shikonin strongly inhibits cell proliferation via modulation of expression of the G1 cell cycle-related proteins and that it induces apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells. Therefore, our data suggest the possibility that shikonin could serve as a novel therapeutic treatment for human tongue squamous cell carcinoma.