Acetylshikonin Induces Apoptosis in Human Colorectal Cancer HCT-15 and LoVo Cells via Nuclear Translocation of FOXO3 and ROS Level Elevation

Acetylshikonin, a naphthoquinone, is a pigment compound derived from Arnebia sp., which is known for its anti-inflammatory potential. However, its anticarcinogenic effect has not been well investigated. Thus, in this study, we focused on investigating its apoptotic effects against HCT-15 and LoVo cells, which are human colorectal cancer cells. MTT assay, cell counting assay, and colony formation assay have shown acetylshikonin treatment induced cytotoxic and antiproliferative effects against colorectal cancer cells in a dose- and time-dependent manner. DNA fragmentation was observed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Also, the increase of subG1 phase in cell cycle arrest assay and early/late apoptotic rates in annexin V/propidium iodide (PI) double staining assay was observed, which indicates an apoptotic potential of acetylshikonin against colorectal cancer cells. 2′,7′-Dichlorofluorescin diacetate (DCF-DA) staining was used to evaluate reactive oxygen species (ROS) generation in acetylshikonin-treated colorectal cancer cells. Fluorescence-activated cell sorting (FACS) analysis showed that acetylshikonin induced an increase in reactive oxygen species (ROS) levels and apoptotic rate in a dose- and time-dependent manner in HCT-15 and LoVo cells. In contrast, cotreatment with N-acetyl cysteine (NAC) has reduced ROS generation and antiproliferative effects in colorectal cancer cells. Western blotting analysis showed that acetylshikonin treatment induced increase of cleaved PARP, γH2AX, FOXO3, Bax, Bim, Bad, p21, p27, and active forms of caspase-3, caspase-7, caspase-9, caspase-6, and caspase-8 protein levels, while those of inactive forms were decreased. Also, the expressions of pAkt, Bcl-2, Bcl-xL, peroxiredoxin, and thioredoxin 1 were decreased. Furthermore, western blotting analysis of cytoplasmic and nuclear fractionated proteins showed that acetylshikonin treatment induced the nuclear translocation of FOXO3, which might result from DNA damage by the increased intracellular ROS level. This study represents apoptotic potential of acetylshikonin against colorectal cancer cells via translocation of FOXO3 to the nucleus and upregulation of ROS generation.


Introduction
Colorectal cancer is a malignancy that occurs in the colon or the rectum. Both forms may simply form as independent colon or rectal cancers, but they often merge due to their common features [1]. It was reported that colorectal cancer is the third leading cause of cancer death and fourth most common cancer diagnosed in the world [2]. Also, colorectal cancer is the second most leading cause of cancer death in the United States, when men and women are combined.
The incidence of colorectal cancer has been increasing worldwide, remarkably in developing countries [3]. It was suggested that age, genetic, and environmental factors are the main factors on development of colorectal cancer. Wellknown risk factors of colorectal cancer include obesity, red meat and processed meat, smoking habit, alcohol consumption, history of abdominal radiation, and familiar histories [4]. There are also protective factors that have correlation with decreasing colorectal cancer incidence. Regular physical activity and intake of more fruits, vegetables, and high-fiber  Oxidative Medicine and Cellular Longevity diet had evidently reduced development of colorectal cancer after diagnosis [5]. Nowadays, colorectal cancer is transformed to increasingly curable disease due to advanced diagnostics such as routine colonoscopies [6,7]. However, surgical resection remains as the only option to cure colon and rectal cancers [8]. Therefore, identifying the candidate matter as a curative option for colorectal cancer is of high value. Acetylshikonin is one of the naturally produced shikonin derivatives, which are pigment components originated from Lithospermum erythrorhizon roots [9]. Lithospermum erythrorhizon is a Chinese medicinal herb that has various functions such as inhibition of transcription activation in human tumor cells and treatment of wounded skins via promoting inflammatory effects, granulation tissue formation, and inhibition of angiogenesis in vitro and in vivo [10,11]. The effects of acetylshikonin against inflammatory diseases and tumor cells via inhibition of vascular endothelial growth factor-(VEGF-) induced angiogenesis were well investigated formerly in previous studies [12,13]. There were also a number of studies which investigated anticarcinogenic effects of acetylshikonin in human cancer cells [14,15]. However, the apoptotic mechanism, of acetylshikonin against colorectal cancer cells is still not thoroughly investigated.
Forkhead box O-3 (FOXO3) is a member of the human transcript factor forkhead box (FOX) gene family [16], which exhibits associations with longevity in human populations and its genetic polymorphisms [17]. FOXO3 is a regulator protein responsible for oxidative stress resistance, metabolism, cell cycle, and cellular apoptosis [18]. FOXO3 protein levels can be regulated by degradation, transcription, and mutation in FOXO genes [19]. Various protein modification mechanisms are involved in regulation of FOXO3. Downregulation of FOXO3 can be mediated by phosphorylation of FOXO3 protein by protein kinase B (Akt) [20,21] or polyubiquitination led by SIRT1/SIRT2-induced deacetylation of FOXO3 [22]. Conversely, increased transcriptional activity of FOXO3 can be induced by phosphorylation of active protein kinases such as 5 ′ -AMP-activated protein kinase (AMPK), Akt, and glycogen synthase kinase-3 (GSK-3) [23,24]. Increased FOXO3 activity induces apoptotic signaling via facilitating the expressions of proapoptotic Bcl2 family members or stimulation of tumor necrosis factor (TNF) family [25,26]. Recent studies have revealed the association between FOXO3 and cellular apoptosis of tumor cells [26][27][28], which suggest that the further investigation of FOXO3related apoptotic mechanisms in tumor cells will bring about a promising strategy on developing therapeutic alternatives.
In this study, we hypothesized that acetylshikonin induces apoptosis in colorectal cancer via activation of FOXO3 expression, thus the downstream apoptotic pathways. To verify the hypothesis, we performed various apoptosis analyses to detect induction of apoptosis and western

Cell Cycle Arrest.
A cell cycle arrest induced by acetylshikonin in HCT-15 and LoVo cells was analyzed. The cells were collected after 24 and 48 h of treatment with acetylshikonin. Then, the cells were suspended in cold 70% ethanol and fixed at -20°C for 18 h. The fixed cells were centrifuged, and supernatants were carefully removed with a pipette. Pelleted cells were incubated with 1 mL of DNA staining solution (50 μg/mL of propidium iodide and 200 μg/mL of DNase-free RNase in PBS with Triton X-100 diluted to 0.2% for permeability) for 30 min. An FC 500 series cytometer (Beckman Coulter) was used for acquisition and analysis. Flow cytometric data were organized using the CXP program (Beckman Coulter) [33].
HCT-15 and LoVo cells were seeded in 6-well plates at a density of 4 × 10 5 per well and cultured for 24 h. After treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM), cells were fixed with 4% formaldehyde solution for 25 min at 4°C and permeabilized using Triton X-100, diluted to 0.5% in PBS for 10 min. Apoptotic cells were treated with 25 μL TdT enzyme buffer. All cells were then stained using Hoechst stain solution (Sigma, St. Louis, MO, USA). Fluorescence-labeled damaged DNA strands were visualized using a fluorescence microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan). Images were taken at 200x magnification [34].       2.11. Cytoplasmic and Nuclear Protein Fractionation. Cytoplasmic and nuclear proteins were separately extracted using Nuclear/Cytosol Fractionation Kit (K266, BioVision, Inc., Milpitas, CA, USA). After 12 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM), cells were collected and washed 2 times with PBS. Supernatants were removed, and cells were resuspended in cytosol extraction buffer-A (CEB-A), vortexed for 15 s at highest setting and incubated in ice for 10 min. Ice-cold cytosol extraction buffer-B (CEB-B) was then added, vortexed 5 s at highest setting and incubated in ice for 1 min. The samples were vortexed for 5 s and centrifuged (14,000 rpm at 4°C for 5 min) to acquire cytoplasmic fraction. The remaining pellets were washed 2 times with PBS, and nuclear extraction buffer (NEB) was added, vortexed 15 s at highest setting and incubated in ice for 10 min. After the vortex and incubation procedure was repeated four times, the samples were centrifuged (14,000 rpm at 4°C for 10 min) to acquire nuclear fraction. The procedure was performed according to the manufacturer's protocol.
2.12. Statistical Analysis. The results are expressed as the arithmetic mean + standard deviation. To compare the data between the groups, two-sided unpaired Student's t-test was used. Experiments were repeated three times, and the representative data were shown. A one-way ANOVA followed by the Bonferroni post hoc test was used for statistical analysis, and a p value of <0.05 was considered statistically significant.  (Figure 1(a)). Cell counting assay and colony forming assay were performed to analyze and visually demonstrate the effect of acetylshikonin in proliferation of colorectal cancer cells. The result from cell counting assay showed decreases of cell viability in acetylshikonin-treated cells (Figure 1(b)). Furthermore, the results from colony forming assay show that the treatment of acetylshikonin had completely inhibited the proliferation of HCT-15 and LoVo cells even in the lowest concentration at low confluency of colorectal cancer cells (Figure 1(c)). These results indicate that acetylshikonin has an inhibitory effect against the cell viability of human colorectal cancer HCT-15 and LoVo cells in a dose-and timedependent manner.

Acetylshikonin Induced Apoptotic Stimulus in HCT-15
and LoVo Cells. To investigate the apoptotic effect of acetylshikonin against colorectal cancer cells, western blotting was performed. Treatment of acetylshikonin induced cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), caspase-3, caspase-7, caspase-9, caspase-6, and caspase-8, which are important modulators of apoptosis in both HCT-15 and LoVo cells. Also, the expressions of antiapoptotic proteins peroxiredoxin (Prdx), thioredoxin 1 (Trx1), Bcl-2, p-Bcl-2, Bcl-xL, and pBad were downregulated, while expressions of proapoptotic proteins Bim, Bax, and Bad were upregulated. Moreover, the expressions of kinase proteins were altered. In acetylshikonin-treated colorectal cancer cells, the protein levels of phosphorylated mitogen-activated protein kinase (p-ERK, p-JNK, and p-p38) were increased, while protein level of pAkt was decreased. The protein levels of p21, p27, and FOXO3, which are proteins related to inhibition of cell proliferation and survival, were upregulated (Figure 7).  cancer cells had slightly inhibited cleavage of PARP and activation of caspase 3, which are key indicators of apoptosis ( Figure 8). This result shows certain evidence that upregulation of MAPK in HCT-15 and LoVo is correlated to acetylshikonin-induced apoptosis. To investigate the protein location and level of FOXO3 and p27 in acetylshikonintreated colorectal cancer cells, nuclear fractional western blotting was performed. Treatment with acetylshikonin in HCT-15 and LoVo cells for 12 h induced decrease of cytoplasmic protein levels of FOXO3 and p27 and increase of nuclear protein levels of those proteins in a dose-dependent manner (Figure 9). These results suggest that acetylshikonin has triggered the translocation of the p27 and FOXO3 proteins from the cytoplasm into the nucleus in HCT-15 and LoVo cells.

Discussion
Phytochemicals are in considerable interests as source of drug or chemotherapy development options [37,38]. Acetylshikonin is a naturally occurring naphthoquinone, a shikonin derivative, which is noted for its inflammatory and anticancer effects [39]. The recent studies have revealed anticancer and preventive effects of acetylshikonin, such as ROSmediated caspase activation [40], induction of cell cycle arrest via p21 and caspase-3 activation [41], and suppression of the NF-κB activity [42] in various cancer cell lines. However, the anticancer effects of acetylshikonin in colorectal cancer via FOXO3 activation have not been well studied. Thus, in this study, we aimed to reveal antiproliferative effect of acetylshikonin in colorectal cancer cells and to present as an alternative candidate agent. Cell apoptosis can be triggered by intrinsic or extrinsic pathways; intrinsic cues involve changes in mitochondria membrane permeability, and extrinsic cues involve activation of death receptors such as Fas or TNFαR, which both cues lead to caspase activation [43,44]. The result from western blotting analysis shows that acetylshikonin induced activation of both caspase-8 and caspase-9 in HCT-15 and LoVo cells (Figure 7), which are the key initiators of caspase cascades in apoptotic cells [45]. Activation of caspase-8 and caspase-9 led to activation of caspase-3, caspase-6, and caspase-7 and resulted in apoptosis of colorectal cancer cells.
FOXO3 is now in a great interest in clinical research as a prognostic biomarker for cancer patients [46]. Phosphorylation of FOXO3 at Ser184 by Akt provides binding site for 14-3-3 chaperone protein, which results in delocalization from the nucleus and degradation by the ubiquitin-proteasome system [47,48]. It was also reported that at high levels of Akt-mediated phosphorylation of FOXO3, full recovery and survival of cancer patients has a low rate [49]. In contrary, FOXO3 activity can be promoted by stress-activated MAPK (p38, JNK, and pERK) [50]. Phosphorylation of FOXO3 at Ser 574, which is a phosphorylation site of JNK, promotes localization of FOXO3 in the nucleus and transcription [51]. Also, the previous study has shown that expression of ERK and p38 proteins had induced phosphorylation of FOXO3 and increased its activity [52]. Our western blotting results (Figures 7 and 8) show that acetylshikonin treatment induced inhibition of Akt and activation of MAPK, which resulted in activation of FOXO3 protein. Also, inhibition of MAPK inhibited the apoptotic signaling in both colorectal cancer cells (Figure 8). Moreover, the nuclear fractional western blot analysis (Figure 9) has confirmed the translocation of FOXO3 to the nucleus, which leads to upregulation of series of target genes and apoptosis consequently [53]. These results show that our findings are relevant to the previous studies.
We found that nuclear translocation of p27 was also occurred by acetylshikonin treatment (Figure 9), which results in suppression of cell cycle progression and induction of apoptosis. Overexpression of cytoplasmic p27 may inhibit apoptosis in tumor cells, via mediating activation of Akt, which is a canonical suppressor protein of apoptosis, and inhibition of cytochrome c release and caspase activation [54,55]. Despite these properties in the cytoplasm, p27 acts as an inhibitor of CDK2 when localized in the nucleus [56,57]. According to the previous studies, nuclear localization of p27 does not just play a role in cellular apoptosis by itself but also supports nuclear translocation of FOXO3 via inhibiting activation of Akt.
FOXO3 translocation (activation) is also a critically involved in ROS accumulation and ROS-mediated apoptosis. FOXO3 regulates the expression of proapoptotic BH3-only proteins (Bim and Bad) and antiapoptotic Bcl-2 proteins (Bcl-2 and Bcl-xL), which triggers mitochondrial membrane permeabilization [58]. Our western blotting analysis results (Figure 7) show that the expressions of proapoptotic proteins were upregulated, while those of antiapoptotic proteins are downregulated in a dose-dependent manner. Moreover, the protein levels of Prdx and Trx1 were downregulated in acetylshikonin-treated cells. In Trx system, those include Prdx and Trx1 are involved in intracellular redox homeostasis regulation and signaling cascade that mediates apoptosis [59]. NAC is an antioxidant generally used in investigating roles of ROS. It inhibits activation of MAPK (JNK, p38) and several proapoptotic proteins, thus enhances cell survival [60]. Our results analyzed with NAC-treated cells show increase of cell viability in NAC-treated cells (Figure 6), which support the data that acetylshikonin-induced apoptosis is mediated by intracellular ROS accumulation. These results imply that treatment of acetylshikonin induces nuclear translocation of FOXO3 followed by ROS-mediated apoptosis signaling cascade.
In this study, we examined the apoptotic activity of acetylshikonin against HCT-15 and LoVo cells and investigated its apoptotic mechanisms. We have revealed that the apoptotic activity of acetylshikonin in HCT-15 and LoVo cells was mediated by translocation of FOXO3 into the nucleus, which induced by Akt inhibition and activation of MAPK. This results in mitochondrial membrane permeabilization, which leads to activation of caspase cascade and apoptosis consequently ( Figure 10). However, the experiments were done only in cellular level; thus, further studies must be proceeded to elucidate the anticancer effects in vivo. This study is believed to provide general understanding of apoptotic mechanisms of acetylshikonin in colorectal cancer and give