In vitro and in vivo antitumor effects of acetylshikonin isolated from Arnebia euchroma (Royle) Johnst (Ruanzicao) cell suspension cultures

Background Shikonin derivatives have cytotoxic and antitumor effects. This study aims to investigate the antitumor effects of acetylshikonin isolated from a Chinese medicinal herb Arnebia euchroma (Royle) Johnst. Methods The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the in vitro antitumor effects of acetylshikonin on human lung adenocarcinoma cell line A549, human hepatocellular carcinoma cell line Bel-7402, human breast adenocarcinoma cell line MCF-7 and mouse Lewis lung carcinoma (LLC) cell line. C57BL/6 mice with LLC model were used to study the in vivo antitumor effects of acetylshikonin. The expression of bax, bcl-2 and caspase-3 proteins in LLC tissue was determined with immunohistochemical staining. Results In A549, Bel-7402, MCF-7 and LLC cell lines, acetylshikonin inhibited cell growth in a dose-dependent manner. IC50 (means ± SD) were 5.6 ± 0.86 μg/ml, 6.82 ± 1.5 μg/ml, 3.04 ± 0.44 μg/ml and 2.72 ± 0.38 μg/ml respectively. Acetylshikonin suppressed tumor growth in C57BL/6 mice with LLC. The inhibition rate of acetylshikonin (2 mg/kg) was 42.85%. Immunohistochemical staining revealed that in the acetylshikonin groups the expression of bax and caspase-3 increased, whereas the expression of bcl-2 decreased, suggesting that acetylshikonin induced tumor cell apoptosis through activating the pro-apoptotic bcl-2 family and caspase-3. Conclusion Acetylshikonin isolated from Arnebia euchroma (Royle) Johnst cell suspension cultures exhibits specific in vivo and in vitro antitumor effects.


Background
Arnebia euchroma (Royle) Johnst (Ruanzicao), a Chinese medicinal herb that induces apoptosis and exerts antitumor effects, is used to treat inflammatory diseases and cancer [1]. Shikonin derivatives, e.g. shikonin, acetylshikonin ( Figure 1), β, β-dimethyl-acrylshikonin, are active components in Arnebia euchroma (Royle) Johnst. Natural shikonin-like compounds have in vitro inhibitory effects on malignant carcinoma cells. Zhen et al. [2] showed that shikonin induced apoptosis of human malignant melanoma A375-S2 cells via activated p53 and caspase-9 pathways. Yoon et al. [3] found that shikonin induced HL60 cells apoptosis via caspase-3 dependent pathways. Gao et al. [4] reported that shikonin reacted with cellular thiols such as glutathione, and that the depletion of cellular thiols induced apoptosis in HL60 cells. Natural shikonin-like compounds also have significant in vivo antitumor effects. In a study by He et al. [5], SYUNZ-7, a shikonin derivative, showed antitumor effects both in vivo and in vitro. Xie et al. [6] showed that some shikonin derivatives were more powerful than natural shikonin in terms of antitumor effects on EAC and S180. Kim et al. [7] reported that 2-hyim-DMNQ-S33, another shikonin derivative, prolonged the survival time of mice bearing S180.
Due to limited distribution and difficult cultivation of Arnebia euchroma (Royle) Johnst, we used the vegetal cell suspension culture technique for the biosynthesis of shikonin-like compounds. Two compounds, namely acetylshikonin and isobutyrylshikonin, have been isolated from the culture vegetal cell suspension.
This study aims to evaluate the in vivo and in vitro antitumor effects of acetylshikonin extracted from the cell suspension cultures of Arnebia euchroma (Royle) Johnst.

Cell lines and cell culture
Malignant cell lines in this study include human lung adenocarcinoma epithelial cell line A549 (ATCC CCL-185), human breast adenocarcinoma cell line MCF-7 (ATCC HTB-22TM) and mouse Lewis lung carcinoma (LLC) (ATCC CRL-1642) were obtained from American Type Culture Collection (USA). Human hepatocellular carcinoma cell line Bel-7402 was obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (Minhai Bio-engineering, China) and maintained at 37°C with 4% CO 2 in a humidified atmosphere. Cell viability was determined with 0.1% trypan blue.

MTT assay
MTT assay [2] was performed to measure the anti-proliferation effects of acetylshikonin on the cell lines of A549, Bel-7402, MCF-7 and LLC. Acetylshikonin was diluted and added to target cells in triplicates with final concentrations at 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4 μg/ml. The cells were incubated for 48 hours and 20 μl of 5 mg/ml solution of MTT in phosphate-buffered saline (PBS) was added to triplicate samples and the plates were incubated for additional 4 hours. The plates were then centrifuged and the medium was removed. Two hundred microliters (200 μl) of DMSO was added to each well to dissolve the purple blue sediment, the absorbance was determined at 590 nm on a microplate reader (Model 550, Bio-Rad, USA). The inhibition rate was calculated as follows: The 50% inhibitory concentrations (IC 50 ) of the 48 hours were calculated with Bliss assay.

Cell growth curve assay
Similarly, A549 cell was used to observe the effects of acetylshikonin on growth curve at various time points. Acetylshikonin was added to A549 cell with various final concentrations (3.2, 1.6, 0.8 μg/ml). MTT assay was performed on the cells in triplicates for each concentration after the cells were incubated for 12, 24, 48, 72 hours respectively. Adriamycin (0.1 μg/ml) served as a positive control.

Mouse model preparation and treatment
The C 57 BL/6 mice (Experimental Animal Center, West China Center for Medical Sciences, Sichuan University, China) were transplanted with LLC according to protocols of transplanted tumor research. At 24 hours after tumor transplantation, the mice were divided into five groups (12 mice per group) randomly: (1) control group (0.9% normal saline), (2) cyclophosphamide group (60 mg per kg of body weight), (3) acetylshikonin group (0.5 mg per kg of body weight), (4) acetylshikonin group (1 mg per kg of body weight) and (5) acetylshikonin group (2 mg per kg of body weight). Mice in cyclophosphamide group received injections only on Day 1, while mice in all other groups received injections once every two days (six times in total). All injections were carried out intraperitoneally. Data were collected and calculated as follows: (2) Inhibitory rate: The animals were sacrificed on Day 13 and tumors were exercised and weighed. Tumor inhibition rate (%) = (1-tumor tissue weight of treatment group/tumor tissue weight of control group) ×100% The animal handlings and experimental procedures were approved by the Animal Ethics Committee of Sichuan University.

Immunohistochemistry
Tumor tissues were fixed with 4% formaldehyde solution at 4°C for 24 hours, dehydrated in graded concentrations of ethanol embedded in paraffin and sliced.
Streptavidin/biotin-peroxidase (SP) method was used for immunohistochemical staining. The primary antibodies, namely bcl-2, bax and caspase-3 (Wuhan Boster Biological Technology, China), were diluted at 1:100. PBS was used as control. Each slice was photographed and the integrated optical density (IOD) was measured with Image pro plus 5.02 (Media Cybernetics, USA).

Statistical analysis
Data were expressed as mean ± standard deviation (SD) unless otherwise indicated. Statistical differences between the treatment and control groups were determined by Mann-Whitney test with SPSS 12.0 (SPSS, USA). P < 0.05 was considered statistically significant.

In vitro effects of acetylshikonin on tumor cell viability
After treatment with acetylshikonin for 48 hours, the untreated tumor cells grew and the cytoskeletons were clearly visible under inverted light microscope. Cells treated with 25.6 μg/ml acetylshikonin became round in shape and condensed nuclei were seen, many of which lost intact membranes, leading to necrosis (Figure 2).

Effects of acetylshikonin on tumor volume
Since Day 5, significant differences were observed between the control and cyclophosphamide and acetylshikonin groups. Tumor volume of the mice in the cyclophosphamide group increased slowly, suggesting that the growth of LLC was suppressed by cyclophosphamide. Tumor volume of the mice in the acetylshikonin group (2 mg/kg) was markedly smaller than that in the control group, suggesting that the growth of LLC was suppressed by acetylshikonin ( Figure 5).

Inhibitory rate on LLC
Cyclophosphamide and acetylshikonin (1, 2 mg/kg) had significant inhibitory effects on the growth of LLC in mice. Inhibitory rate of the cyclophosphamide group was 67.81%; inhibition rates of the three acetylshikonin groups were 42.85%, 21.86% and 11.11% respectively, all in a dose-dependent manner (Table 1, Figure 6).

Immunohistochemistry evaluation
The photomicrograph of immunohistochemistry staining is shown in Figure 7. The positive reaction located in cytosol was stained in brown. The color of the stain is positively correlated to the protein expression.
The IOD of each group indicates that the expression of bax and caspase-3 in the acetylshikonin groups increased, whereas bcl-2 decreased in the same groups, resulting in higher bax/bcl-2 ratios, all in a dose-dependent manner ( Table 2).

Discussion
Recent studies showed that shikonin derivatives acted on multiple tumor cells and triggered multiple cell death pathways. Therefore, shikonin derivatives are potential cancer treatment agents. Acetylshikonin is a main shikonin derivative of Arnebia euchroma (Royle) Johnst.
had in vitro and in vivo antitumor effects. Acetylshikonin possessed a high level of cytotoxic activity in vitro. The present study showed that tumor volume and weight of the mice treated with acetylshikonin increased more slowly than the control in vivo.
Apoptosis is critical in the development of tumor. Antiapoptotic agents such as bcl-2 are initially integral membrane proteins in mitochondria, endoplasmic reticulum (ER) or nuclear membrane [9]. These agents inhibit apoptosis by regulating Ca 2+ fluxes through ER membrane [10]. Over expression of bcl-2 turns cells suffering from irrevers-ible gene mutation to normal cell cycle rather than apoptosis, thereby causing cancer. In contrast, bax is a proapoptotic molecule that can induce cell apoptosis. In viable cells, bax is either in the cytosol or loosely attached to membranes. In response to a death stimulus, the cytosolic bax translocates to mitochondria where it becomes an integral membrane protein and cross-linkable as homodimers, creating a pathway for cytochrome c to release and activate caspase [11]. As the bcl-2 family proteins act upstream from irreversible cellular damage and all have effects in mitochondria, the ratio of bcl-2 and bax determines whether a cell will live or die [12]. Apoptosis  manifests in two major execution programs downstream from the death signal: the caspase pathway and organelle dysfunction, mitochondrial dysfunction in particular. Caspases play an essential role during apoptotic cell death. There are two relatively well characterized caspase cascades: one is initiated by the activation of cell-surface death receptors, such as Fas and tissue necrosis factor, leading to caspase-8 activation which in turn cleaves and activates downstream caspases; the other is triggered by cytochrome c released from mitochondria, which promotes the activation of caspase-9 and caspase-3, thereby initiating caspase cascade to induce cell apoptosis [13].
Our results suggest that acetylshikonin activates the proapoptotic bcl-2 family, releases cytochrome c and activates caspase-3, thereby inducing tumor cell apoptosis.
Hsu et al. [14] found that shikonin activated caspase and induced apoptosis via modulating bcl-2 family, p27 and p53. Liu et al. [15] found that certain shikonin derivatives (acetylshikonin) act as modulators of the Nur77-mediated apoptotic pathway and identify a new shikoninbased lead that targets Nur77 for apoptosis induction. Xuan and Hu [8] reported that Shikonin derivatives circumvented diverse cancer drug resistance (P-gp, MRP1, BCRP1, Bcl-2, Bcl-xL) by inducing a dominant necrosis. Apart from inducing apoptosis, acetylshikonin may also inhibit DNA topoisomerase, reduce carcinogenesis and possess antimitogenic and angiogenic actions [16,17]. As a possible wide spectrum agent combating cancer through various mechanisms, acetylshikonin may be a therapeutic candidate.