Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.40 No.1 pp.51-61
DOI : https://doi.org/10.11620/IJOB.2015.40.1.051

Mechanism Underlying Shikonin-induced Apoptosis and Cell Cycle Arrest on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

Sang-Hun Oh, Sung-Jin Park, Su-Bin Yu, Yong-Ho Kim, In-Ryoung Kim, Bong-Soo Park*
Department of Oral Anatomy, School of Dentistry, Pusan National University
Corresponding Author : Bong-Soo Park, Department of Oral Anatomy, School of Dentistry, Pusan National University, Yangsan, 626-870, Korea. Tel.: +82-51-510-8242, Fax: +82-51-510-8241 parkbs@pusan.ac.kr
March 2, 2015 March 17, 2015 March 17, 2015

Abstract

Shikonin, a major ingredient in the traditional Chinese herb Lithospermumerythrorhizon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. It has recently been reported that shikonin displays antitumor properties in many cancers. This study was aimed to investigate whether shikonin could inhibit oral squamous carcinoma cell (OSCC) growth via mechanisms of apoptosis and cell cycle arrest. The effects of shikonin on the viability and growth of OSCC cell line, SCC25 cells were assessed by MTT assay and clonogenic assays, respectively. Hoechst staining and DNA electrophoresis indicated that the shikonin-treated SCC25 cells were undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, flow cytometry, MMP activity, and proteasome activity also supported the finding that shikonin induces apoptosis. Shikonin treatment of SCC25 cells resulted in a time- and dose-dependent decrease in cell viability, inhibition of cell growth, and increase in apoptotic cell death. The treated SCC25 cells showed several lines of apoptotic manifestation as follows: nuclear condensation; DNA fragmentation; reduced MMP and proteasome activity; decrease in DNA contents; release of cytochrome c into cytosol; translocation of AIF and DFF40 (CAD) onto the nuclei; a significant shift in Bax/Bcl-2 ratio; and activation of caspase-9, -7, -6, and -3, as well as PARP, lamin A/C, and DFF45 (ICAD). Shikonin treatment also resulted in down-regulation of the G1 cell cycle-related proteins and up-regulation of p27KIP1. Taken together, our present findings demonstrate that shikonin strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins, and that it induces apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells.


초록


    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), p27KIP1, 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 p27KIP1was 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 p21WAF1/CIP1 and p27KIP1 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 p27KIP1 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, p27KIP1 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.

    Conflict of interest

    The authors declare that they have no conflicting interest.

    Figure

    IJOB-40-51_F1.gif

    Shikonin treatment had the effect of decreasing SCC25 cell viability. Cells treated with shikonin (0-10 μM) for 24–72 hours showed a remarkable reduction in viability in both a dose- and time-dependent manner.

    IJOB-40-51_F2.gif

    The effect of growth inhibition on SCC25 cells was examined by clonogenic assay. SCC25 cells were cultured in the presence of the indicated concentrations (0–2.5 μM) of shikonin for seven days. (A) Photograph showing colony formation in SCC25 cells. (B) The growth of the shikonin-treated groups is expressed as percentage of the control. Note that shikonin significantly inhibited the growth of SCC25 cells. Values are means ± standard deviation of triplicates of independent experiments.

    IJOB-40-51_F3.gif

    Demonstration of apoptosis in SCC25 cells treated with shikonin for 24 hours. (A) Immunofluorescence micrographs after Hoechst staining. Cells not treated with shikonin display round nuclei (0 μM). Cells treated with shikonin (2.5–10 μM) for 24 hours showed the production of nuclear condensation, which increased in a dose-dependent manner. (B) The values shown below the micrographs are the means ± standard deviation of the apoptotic cells as determined by Hoechst staining.

    IJOB-40-51_F4.gif

    DNA fragmentation was demonstrated by DNA electrophoresis. The 5-10 μM shikonin treatment groups clearly showed DNA degradation characteristic of apoptosis with a ladder pattern of DNA fragments. The SCC25 cells were treated with shikonin for 24 hours.

    IJOB-40-51_F5.gif

    Shikonin-induced apoptosis increased in a dosedependent manner. The cells were treated with shikonin (1-10 μ M) for 24 hours, and the ratio of apoptotic cells was determined by fluorescence-activated cell sorting flow cytometry analysis.

    IJOB-40-51_F6.gif

    Bcl-2 and Bax expression levels in SCC25 cells treated with shikonin were detected by Western blot analysis. Bax, a proapoptotic factor, increased significantly in a dose-dependent manner, whereas Bcl-2, an antiapoptotic factor, decreased. GAPDH was used as a loading control for quantifying Bcl-2 and Bax expression.

    IJOB-40-51_F7.gif

    Mitochondrial membrane potential (△Ψm) at 24 hours after 1-10 μM shikonin treatment. △Ψm decreased in SCC25 cells in a dose-dependent manner. △Ψm was measured by flow cytometry.

    IJOB-40-51_F8.gif

    Confocal microscopy showed that cytochrome c was released from mitochondria into the cytoplasm in SCC25 cells treated with 5 μM shikonin.

    IJOB-40-51_F9.gif

    Apoptosis-inducing factor (AIF) was released from the mitochondria into the cytoplasm and then translocated onto the nucleus in SCC25 cells treated with 5 μM shikonin. Confocal microscopy images show the colocalization of AIF and nuclei.

    IJOB-40-51_F10.gif

    Western blot analyses of caspase-9, caspase-7, caspase-3, PARP, caspase-6, lamin A/C and DFF45 (ICAD) in SCC25-treated cells. Shikonin treatment induced caspase-9, caspase-7, caspase-3, PARP, caspase-6 and lamin A/C degradation and produced processed caspase-7 20 kDa, caspase-3 17 kDa, PARP 85 kDa, caspase-6 17 kDa and lamin A/C 65 kDa cleaved products, repectively. Also, cleaved form of DFF45 (ICAD) (34 and 11 kDa) was activated by shikonin treatment. GAPDH levels were used as a loading standard for quantifying protein expression.

    IJOB-40-51_F11.gif

    DFF40 (CAD) translocated from cytosol into the nuclei. (A) Confocal microscopy images showing the colocalization of CAD and nuclei. Enhanced colocalization is seen in the Shikonin-treated group (b). (B) Depiction of CAD profile and nuclei fluorescence intensity. CAD intensity is shown in green, and nucleus intensity is shown in blue. The green and blue intensities overlap in the shikonin-treated group.

    IJOB-40-51_F12.gif

    Effects of shikonin treatment on SCC25 cell cycle progression. (A) Representative DNA histograms. Shikonin treatment significantly increased the number of apoptotic cells with DNA hypoploidy in a time-dependent manner. (B) The percentages of G1, S, and G2 are depicted. Data shown are representative of three independent experiments.

    IJOB-40-51_F13.gif

    Cell cycle-related proteins were activated by shikonin. p27KIP1 levels increased remarkably, whereas Cdk2, Cdk4, Cyclin D1, and Cyclin D3 levels decreased in a time-dependent manner. GAPDH levels were used as a housekeeping gene for quantifying p27KIP1, Cdk2, Cdk4, Cyclin D1, and Cyclin D3 expression.

    Table

    Reference

    1. Andujar I , Recio MC , Giner RM , Rios JL (2013) Traditional chinese medicine remedy to jury: the pharmacological basis for the use of shikonin as an anticancer therapy , Curr Med Chem, Vol.20; pp.2892-2898http://dx.doi.org/10.2174/09298673113209990008
    2. Andujar I , Rios JL , Giner RM , Recio MC (2013) Pharmacological Properties of Shikonin - A Review of Literature since 2002 , Planta Med,
    3. Zhang FL , Wang P , Liu YH , Liu LB , Liu XB , Li Z , Xue YX (2013) Topoisomerase I inhibitors, shikonin and topotecan, inhibit growth and induce apoptosis of glioma cells and glioma stem cells , PLoS One, Vol.8; pp.e81815
    4. Piao JL , Cui ZG , Furusawa Y , Ahmed K , Rehman MU , Tabuchi Y , Kadowaki M , Kondo T (2013) The molecular mechanisms and gene expression profiling for shikonininduced apoptotic and necroptotic cell death in U937 cells , Chem Biol Interact, Vol.205; pp.119-127http://dx.doi.org/10.1016/j.cbi.2013.06.011
    5. Rajasekar S , Park da J , Park C , Park S , Park YH , Kim ST , Choi YH , Choi YW (2012) In vitro and in vivo anticancer effects of Lithospermum erythrorhizon extract on B16F10 murine melanoma , J Ethnopharmacol, Vol.144; pp.335-345
    6. Wang HB , Ma XQ (2012) [Mechanisms of (2-methyl-n-butyl) shikonin induced apoptosis of gastric cancer SGC-7901 cells] , Yao Xue Xue Bao, Vol.47; pp.816-821
    7. Yeh CC , Kuo HM , Li TM , Lin JP , Yu FS , Lu HF , Chung JG , Yang JS (2007) Shikonin-induced apoptosis involves caspase-3 activity in a human bladder cancer cell line (T24) , In Vivo, Vol.21; pp.1011-1019
    8. Zhang Z , Zhang C , Ding Y , Zhao Q , Yang L , Ling J , Liu L , Ji H , Zhang Y (2014) The activation of p38 and JNK by ROS, contribute to OLO-2-mediated intrinsic apoptosis in human hepatocellular carcinoma cells , Food Chem Toxicol, Vol.63; pp.38-47http://dx.doi.org/10.1016/j.fct.2013.10.043
    9. Carson DA , Ribeiro JM (1993) Apoptosis and disease , Lancet, Vol.341; pp.1251-1254
    10. Ohta K , Yamashita N (1999) Apoptosis of eosinophils and lymphocytes in allergic inflammation , J Allergy Clin Immunol, Vol.104; pp.14-21
    11. Elmore S (2007) Apoptosis: a review of programmed cell death , Toxicol Pathol, Vol.35; pp.495-516
    12. Fulda S , Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy , Oncogene, Vol.25; pp.4798-4811
    13. Chang KP , Wu CC , Fang KH , Tsai CY , Chang YL , Liu SC , Kao HK (2013) Serum levels of chemokine (C-X-C motif) ligand 9 (CXCL9):are associated with tumor progression and treatment outcome in patients with oral cavity squamous cell carcinoma , Oral Oncol, Vol.49; pp.802-807http://dx.doi.org/10.1016/j.oraloncology.2013.05.006
    14. Chen SF , Nieh S , Jao SW , Liu CL , Wu CH , Chang YC , Yang CY , Lin YS (2012) Quercetin suppresses drug-resistant spheres via the p38 MAPK-Hsp27 apoptotic pathway in oral cancer cells , PLoS One, Vol.7; pp.e49275
    15. Quan J , Elhousiny M , Johnson NW , Gao J (2013) Transforming growth factor-beta1 treatment of oral cancer induces epithelial-mesenchymal transition and promotes bone invasion via enhanced activity of osteoclasts , Clin Exp Metastasis, Vol.30; pp.659-670
    16. Bell RB , Kademani D , Homer L , Dierks EJ , Potter BE (2007) Tongue cancer: Is there a difference in survival compared with other subsites in the oral cavity? , J Oral Maxillofac Surg, Vol.65; pp.229-236
    17. Lo WL , Kao SY , Chi LY , Wong YK , Chang RC (2003) Outcomes of oral squamous cell carcinoma in Taiwan after surgical therapy: factors affecting survival , J Oral Maxillofac Surg, Vol.61; pp.751-758
    18. Shintani S , Li C , Mihara M , Klosek SK , Terakado N , Hino S , Hamakawa H (2006) Anti-tumor effect of radiation response by combined treatment with angiogenesis inhibitor, TNP-470, in oral squamous cell carcinoma , Oral Oncol, Vol.42; pp.66-72
    19. Shen J , Huang C , Jiang L , Gao F , Wang Z , Zhang Y , Bai J , Zhou H , Chen Q (2007) Enhancement of cisplatin induced apoptosis by suberoylanilide hydroxamic acid in human oral squamous cell carcinoma cell lines , Biochem Pharmacol, Vol.73; pp.1901-1909
    20. Lachumy SJ , Oon CE , Deivanai S , Saravanan D , Vijayarathna S , Choong YS , Yeng C , Latha LY , Sasidharan S (2013) Herbal Remedies for Combating Irradiation: a Green Antiirradiation Approach , Asian Pac J Cancer Prev, Vol.14; pp.5553-5565http://dx.doi.org/10.7314/APJCP.2013.14.10.5553
    21. Yang Y , Zhang Z , Li S , Ye X , Li X , He K (2013) Synergy effects of herb extracts: Pharmacokinetics and pharmacodynamics basis , Fitoterapia, Vol.92C; pp.133-147http://dx.doi.org/10.1016/j.fitote.2013.10.010
    22. Kim HM , Lee EH , Hong SH , Song HJ , Shin MK , Kim SH , Shin TY (1998) Effect of Syzygium aromaticum extract on immediate hypersensitivity in rats , J Ethnopharmacol, Vol.60; pp.125-131
    23. Kim HM , Yi JM , Lim KS (1999) Magnoliae flos inhibits mast cell-dependent immediate-type allergic reactions , Pharmacol Res, Vol.39; pp.107-111
    24. Park HI , Jeong MH , Lim YJ , Park BS , Kim GC , Lee YM , Kim HM , Yoo KS , Yoo YH (2001) Szygium aromaticum (L.) Merr. Et Perry (Myrtaceae) flower bud induces apoptosis of p815 mastocytoma cell line , Life Sci, Vol.69; pp.553-566
    25. Na HJ , Jeong HJ , Bae H , Kim YB , Park ST , Yun YG , Kim HM (2002) Tongkyutang inhibits mast cell-dependent allergic reactions and inflammatory cytokines secretion , Clin Chim Acta, Vol.319; pp.35-41
    26. Kim JM , Bae HR , Park BS , Lee JM , Ahn HB , Rho JH , Yoo KW , Park WC , Rho SH , Yoon HS , Yoo YH (2003) Early mitochondrial hyperpolarization and intracellular alkalinization in lactacystin-induced apoptosis of retinal pigment epithelial cells , J Pharmacol Exp Ther, Vol.305; pp.474-481
    27. Chen YY , Wang Z (2012) [Advance in study on pharmacological mechanisms of Qingkailing injection in intervention of ischemic cerebral injury] , Zhongguo Zhong Yao Za Zhi, Vol.37; pp.3198-3202
    28. Gu Y , Scheuer C , Feng D , Menger MD , Laschke MW (2013) Inhibition of angiogenesis a novel antitumor mechanism of the herbal compound arctigenin , Anticancer Drugs, Vol.24; pp.781-791
    29. Liu C , Yin L , Chen J (2013) The apoptotic effect of shikonin on human papillary thyroid carcinoma cells through mitochondrial pathway , Tumour Biol,
    30. Wang L , Gai P , Xu R , Zheng Y , Lv S , Li Y , Liu S (2015) Shikonin protects chondrocytes from interleukin-1beta-induced apoptosis by regulating PI3K/Akt signaling pathway , Int J Clin Exp Pathol, Vol.8; pp.298-308
    31. Wyllie AH , Kerr JF , Currie AR (1980) Cell death the significance of apoptosis , Int Rev Cytol, Vol.68; pp.251-306
    32. Williams GT (1991) Programmed cell death apoptosis and oncogenesis , Cell, Vol.65; pp.1097-1098
    33. Kroemer G (2001) [Mitochondrial control of apoptosis] , Bull Acad Natl Med, Vol.185; pp.1135-1142
    34. Green DR , Reed JC (1998) Mitochondria and apoptosis , Science, Vol.281; pp.1309-1312
    35. Susin SA , Lorenzo HK , Zamzami N , Marzo I , Snow BE , Brothers GM , Mangion J , Jacotot E , Costantini P , Loeffler M , Larochette N , Goodlett DR , Aebersold R , Siderovski DP , Penninger JM , Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor , Nature, Vol.397; pp.441-446
    36. Gogvadze V , Orrenius S (2006) Mitochondrial regulation of apoptotic cell death , Chem Biol Interact, Vol.163; pp.4-14
    37. Orrenius S (2004) Mitochondrial regulation of apoptotic cell death , Toxicol Lett, Vol.149; pp.19-23
    38. Narita M , Shimizu S , Ito T , Chittenden T , Lutz RJ , Matsuda H , Tsujimoto Y (1998) Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria , Proc Natl Acad Sci USA, Vol.95; pp.14681-14686
    39. Zou H , Li Y , Liu X , Wang X (1999) An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9 , J Biol Chem, Vol.274; pp.11549-11556
    40. Daugas E , Susin SA , Zamzami N , Ferri KF , Irinopoulou T , Larochette N , Prevost MC , Leber B , Andrews D , Penninger J , Kroemer G (2000) Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis , FASEB. J, Vol.14; pp.729-739
    41. Vaux DL , Silke J (2005) IAPs, RINGs and ubiquitylation , Nat Rev Mol Cell Biol, Vol.6; pp.287-297
    42. Morizane Y , Honda R , Fukami K , Yasuda H (2005) X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO , J Biochem, Vol.137; pp.125-132
    43. Neutzner A , Li S , Xu S , Karbowski M (2012) The ubiquitin/ proteasome system-dependent control of mitochondrial steps in apoptosis , Semin Cell Dev Biol, Vol.23; pp.499-508http://dx.doi.org/10.1016/j.semcdb.2012.03.019
    44. McIlwain DR , Berger T , Mak TW (2013) Caspase functions in cell death and disease , Cold Spring Harb Perspect Biol, Vol.5; pp.a008656
    45. Cheng AC , Jian CB , Huang YT , Lai CS , Hsu PC , Pan MH (2007) Induction of apoptosis by Uncaria tomentosa through reactive oxygen species production, cytochrome c release, and caspases activation in human leukemia cells , Food Chem Toxicol, Vol.45; pp.2206-2218
    46. Pavletich NP (1999) Mechanisms of cyclin-dependent kinase regulation structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors , J Mol Biol, Vol.287; pp.821-828
    47. Polyak K , Lee MH , Erdjument-Bromage H , Koff A , Roberts JM , Tempst P , Massague J (1994) Cloning of p27Kip1, a cyclindependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals , Cell, Vol.78; pp.59-66
    48. Coats S , Flanagan WM , Nourse J , Roberts JM (1996) Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle , Science, Vol.272; pp.877-880
    49. Teyssier F , Bay JO , Dionet C , Verrelle P (1999) [Cell cycle regulation after exposure to ionizing radiation] , Bull Cancer, Vol.86; pp.345-357
    50. Colman MS , Afshari CA , Barrett JC (2000) Regulation of p53 stability and activity in response to genotoxic stress , Mutat Res, Vol.462; pp.179-188