β,β-Dimethylacrylshikonin Induces Mitochondria Dependent Apoptosis through ERK Pathway in Human Gastric Cancer SGC-7901 Cells

β,β-Dimethylacrylshikonin, one of the active components in the root extracts of Lithospermum erythrorhizon, posses antitumor activity. In this study, we discussed the molecular mechanisms of β,β-dimethylacrylshikonin in the apoptosis of SGC-7901 cells. β,β-Dimethylacrylshikonin reduced the cell viability of SGC-7901 cells in a dose- and time-dependent manner and induced cell apoptosis. β,β-Dimethylacrylshikonin treatment in SGC-7901 cells down-regulated the expression of XIAP, cIAP-2, and Bcl-2 and up-regulated the expression of Bak and Bax and caused the loss of mitochondrial membrane potential and release of cytochrome c. Additionally, β,β-dimethylacrylshikonin treatment led to activation of caspases-9, 8 and 3, and cleavage of poly (ADP-ribose) polymerase (PARP), which was abolished by pretreatment with the pan-caspase inhibitor Z-VAD-FMK. β,β-Dimethylacrylshikonin induced phosphorylation of extracellular signal-regulated kinase (ERK) in SGC-7901 cells. U0126, a specific MEK inhibitor, blocked the ERK activation by β,β-dimethylacrylshikonin and abrogated β,β-dimethylacrylshikonin -induced apoptosis. Our results demonstrated that β,β-dimethylacrylshikonin inhibited growth of gastric cancer SGC-7901 cells by inducing ERK signaling pathway, and provided a clue for preclinical and clinical evaluation of β,β-dimethylacrylshikonin for gastric cancer therapy.


Introduction
Gastric cancer is one of the aggressive malignant tumors, although with the development of radiotherapy, chemotherapy and biotherapy, the severe side effects are unavoidable [1], therefore, more effective antitumor drugs with fewer side effects for the treatment of gastric cancer are needed.
Lithospermum erythrorhizon is an important Chinese herb. It has some active components: Deoxyshikonin, Isobutyrylshikonin, Acetylshikonin, Isovalerylshikonin, b,b-Dimethylaerylshikonin (Fig. 1A) and shikonin [2]. In traditional Chinese medicine, it exhibits multiple biological functions including anti-microbial, anti-inflammatory, anti-tumor, immune regulation and anti-HIV properties [3][4][5][6]. The anti-tumor effect of shikonin and its derivatives were first proved by their activities against tumor growth in murine Sarcoma-180 [7]. Shikonin exhibits effect not merely by killing tumor cells directly, but also by inhibiting tumor angiogenesis [8]. Studies revealed that shikonin induced apoptosis in human malignant melanoma, bladder cancer, cervical cancer, lung cancer and liver cancer, and so on [9][10][11][12][13]. However, it has less side effects and more protective effects on human normal cells [14,15]. Hsu PC et al showed that shikonin led to cell apoptosis through up-regulation of p27, p53, Bax and down-regulation of Bcl-2 and Bcl-xL in human colorectal carcinoma COLO 205 cells [16]. The tumor invasion inhibited by shikonin in some cancer cells may through the downregulation of NF-kB-mediated MMP-9 expression [17]. Shikonin also induces apoptosis via ROS production in hepatocellular carcinoma [12]. Singh F et al also found that Shikonin decreased phosphorylated levels of EGFR, ERK and protein tyrosine kinases and increased intracellular levels of apoptosis-related proteins, which caused epidermoid carcinoma cells to undergo apoptosis [18].
Recent evidence showed that b,b-dimethylacrylshikonin had significant anti-tumor effect on hepatocellular carcinoma by activating caspase-3 [19]. However, such effect of b,b-dimethylacrylshikonin on human gastric cancer cells has not been reported and the molecular mechanisms are still not well understood. Thus, in the present study, We will discuss effect of b,b-dimethylacrylshikonin on human gastric cancer cell SGC-7901 and its related signaling to better understand the mechanism of b,b-dimethylacrylshikonin on gastric cancer.
Cell culture and cell proliferation assay SGC-7901 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (Hyclone, UT), and maintained at 37uC in a humidified atmosphere of 5% CO 2 . Then cells were seeded in a 96-well dish to a final concentration of 5610 3 cells/well and incubated in RPMI 1640 medium containing 10% FCS for 24 h, after that, cells were treated with the indicated concentration of b,b-dimethylacrylshikonin for 24 h and 48 h. Medium was removed and fresh medium was added to each well along with 20 ml of MTT solution (5 mg/ml). After 4 h incubation, 150 ml of DMSO was added to each well. The plates were read at wavelength of 570 nm using Varioskan Flash Multimode Reader (Thermo Scientific, USA). Four reduplicate wells were used for each treatment, and experiments were repeated three times.

Morphological changes
SGC-7901 cells were placed in the well of a six-well plate. After 24 h cell culture, they were treated with b,b-dimethylacrylshikonin for the indicated time periods. The cellular morphology was observed using an inverted microscopy (model IX70; Olympus, Tokyo, Japan).
DAPI (49, 6-diamidino-2-phenylindole) staining SGC-7901 cells were placed in the well of a six-well plate. After 24 h cell culture, they were treated with b,b-dimethylacrylshikonin for the indicated time periods, then cells were fixed in cold acetone for 30 min, and incubated with DAPI (1 mg/ml) for 30 min. The apoptotic nuclei characterized as intensively stained were detected using fluorescent microscopy (model IX71; Olympus, Tokyo, Japan).

Annexin V/PI assays for apoptosis
For Annexin V/PI assays, cells were stained with AnnexinV-FITC and PI, and evaluated for apoptosis by flow cytometry according to the mannufacturer's protocol (BD PharMingen, San Diego, CA, USA). Briefly, 1610 5 cells were washed twice with PBS, and stained with 5 ml of AnnexinV-FITC and 5 ml of PI in 500 ml binding buffer for 15 min at room temperature in the dark. The apoptotic cells were determined using BD FACS Diva software (BD Biosciences, Franklin Lakes, NJ). Annexin V staining serves as a measure of phosphatidylserine externalization and cells that are Annexin V + /PI 2 represent early apoptotic cells.

Western Bloting analysis
Cells were treated with the indicated concentration of b,bdimethylacrylshikonin for the indicated time in RPMI 1640 medium with 10% FCS. The cells were collected in ice-cold PBS, and the cell extracts were prepared in RIPA buffer with proteinase inhibitor cocktail from Calbiochem (San Diego, CA). The protein concentrations of the cell lysates were determined and boiled with gel-loading buffer for 10 min at 100uC. Samples containing 30 mg of total protein were electrophoresed on 10% SDS-polyacrylamide gels and transferred to PVDF membrane (Millipore, Temecula, CA). Following transfer, the membrane were blocked in TBST (TBS containing 0.1% Tween 20) containing 5% skimmed milk for 2 h, followed by incubation overnight at 4uC with appropriate primary antibodies. After washing three times in TBST, the membranes were incubated for 2 h at 37uC with 1:5000 horseradish peroxidase-conjugated appropriate secondary antibodies. Finally, the membranes were visualized using the enhanced chemiluminescence detection system (Immun-Star WesternC Kit, Bio-Rad, USA).

Immunofluorescence staining
Immunofluorescene staining was used to analyze the subcellular distribution of cytochrome c in SGC-7901 cells induced by b,bdimethylacrylshikonin. Cells cultured on sterile glass coverlips were fixed in cold acetone for 10 min. After permeabilized with 0.3% Triton X-100 for 20 min at room temperature, cells were blocked in 3% bovine serum albumin for 30 min and incubated overnight at 4uC with anti-cytochrome c antibody(1:30). After washing three times in PBS, SGC-7901 cells were labeled with Alexa Fluor 488-conjugated secondary antibody. DAPI was subsequently added for nuclear staining. Microscopic analysis was performed using a fluorescent microscopy (model IX71; Olympus, Tokyo, Japan).

Statistical analysis
Data reported are the mean6standard deviation (SD) of three independent experiments. They were evaluated by one-way analysis of variance (ANOVA). Significant differences were established at p,0.05.  Fig. 2A). b,bdimethylacrylshikonin also induced nuclear condensation with intensive DAPI staining compared to the nuclear of control SGC-7901 cells for 24 h (Fig. 2B).

b,b-dimethylacrylshikonin induces mitochondrial events associated with apoptosis in SGC-7901 cells
Bcl-2 family proteins include pro-apoptotic proteins (Bax, Bak and Bid) and anti-apoptotic proteins (Bcl-2, Bcl-xL, cIAP-2, XIAP and survivin). They can activate or inhibit the release of downstream factors which lead to the activation of caspase-3 and PARP in the execution of apoptosis [20]. In order to detect the apoptosis-related proteins, pro-apoptotic proteins (Bax, Bak) and anti-apoptotic proteins (XIAP, cIAP-2, Bcl-2, Bcl-xL, survivin) were detected by Western blotting after SGC-7901 cells were treated with different concentration of b,b-dimethylacrylshikonin (0, 2.5, 5, 7.5, and 10 mmol/L) for 24 h. The results indicated that b,b-dimethylacrylshikonin decrease the expression of XIAP, cIAP-2, Bcl-2 (Fig. 3A). However, the down-regulation of XIAP and Bcl-2 were less pronounced while the down-regulation of cIAP-2 was quite dramatic and dose-dependent. There was no significant change of Bcl-xL and survivn in SGC-7901 cells. Whether b,bdimethylacrylshikonin can modulate the expression of proapoptotic proteins (Bax, Bak) was also examined. We found that b,b-dimethylacrylshikonin increased the expression of Bak in a dose-dependent manner but had little effect on Bax (Fig. 3B).
Caspase family proteins are critical enzymes to execute apoptosis. Among caspase family members, caspase-3 is a key executioner for apoptosis in mammalian cells [21]. It can be activated through death receptor-mediated extrinsic caspase-8 pathway and the mitochondria dependent-caspase-9 intrinsic pathway [22,23]. In order to further understand the molecular mechanism of the apoptosis, SGC-7901 cells were treated with b,b-dimethylacrylshikonin and detected by Western blotting analysis for the expression change of caspase-3, caspase-8 and caspase-9, results showed a dose-dependent elevation of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 in b,bdimethylacrylshikonin treated cells. PARP cleavage, another well-known characteristic of apoptosis, was also found in b,bdimethylacrylshikonin treated cells (Fig. 3C). Mitochondrial dysfunction induced apoptosis which is often the consequence of a decrease of mitochondrial membrane potential. It has been shown to be central to the apoptotic pathway. The SGC-7901 cells were treated with or without b,b-dimethylacrylshikonin (10 mmol/L) for 24 h, results showed mitochondria membrane potential was decreased from 98.6% to 56.9% after b,b-dimethylacrylshikonin was treated to the SGC-7901 cells (Fig. 3D). We next examined the distribution and subcellular localization of cytochrome c to find whether the mitochondria pathway is involved in b,bdimethylacrylshikonin induced apoptosis. After SGC-7901 cells were treated with b,b-dimethylacrylshikonin (10 mmol/L) for 24 h, the distribution of cytochrome c was visualized with a confocal laser microscope, the b,b-dimethylacrylshikonin treated cells showed blurred morphology (Fig. 3E) in contrast to the obviously clear appearance in the control cells, indicating the translocation of cytochrome c from the mitochondria into the cytoplasm in b,b-dimethylacrylshikonin treated cells.
To further determine the role of caspase activation in b,bdimethylacrylshikonin-induced apoptosis, we treated SGC-7901 cells with pan-caspase inhibitor Z-VAD-FMK(10 mmol/L) before b,b-dimethylacrylshikonin treatment. The pan-caspase inhibitor Z-VAD-FMK pretreatment decreased the expression of cleaved caspase-3, cleaved PARP and reduced b,b-dimethylacrylshikonininduced apoptosis (Fig. 3F & G). These data showed that mitochondrial-mediated caspase cascade pathway plays a very important role in b,b-dimethylacrylshikonin-induced apoptosis.

b,b-Dimethylacrylshikonin induces sustained ERK activation in SGC-7901 cells
It was shown that MAPK signaling involved in several events of cellular stresses and stimuli induced cell apoptosis [24,25]. We therefore examined the activation of ERK, JNK and p38 after b,b-dimethylacrylshikonin treatment. As shown in Fig. 4A & B, the phosphorylation levels of ERK and JNK were apparently increased in response to the b,b-dimethylacrylshikonin treatment for 2 h, and the phosphorylation levels exhibited a dose-dependent manner. Besides, the phosphorylation levels of ERK last twentyfour hours. However, no significant changes of phosphorylation levels of p38 were observed after the b,b-dimethylacrylshikonin treatment (Fig. 4C). These results suggested that sustained activation of the ERK is involved in b,b-dimethylacrylshikonininduced apoptosis in SGC-7901 cells.

The ERK signaling pathway is involved in b,bdimethylacrylshikonin induced apoptosis in SGC-7901 cells
We then examined whether the b,b-dimethylacrylshikonininduced sustained activation of the ERK signaling pathway plays a role in apoptosis. As shown in Fig. 5A & B, Western blot assay revealed that U0126 (a specific MEK inhibitor) inhibited sustained ERK activation and cleavage of caspase-3, PARP in b,bdimethylacrylshikonin treatment while the pan-caspase inhibitor Z-VAD-FMK had no effect on b,b-dimethylacrylshikonin-induced activation of ERK (Fig. 5D). In addition, U0126 reduced b,bdimethylacrylshikonin-induced apoptosis in SGC-7901 cells (Fig. 5C). These results showed that b,b-dimethylacrylshikonininduced apoptosis in SGC-7901 cells are mediated by the sustained activation of the ERK signaling pathway.

Discussion
Shikonin derivatives are the active components isolated from the Chinese herbal Lithospermum erythrorhizon [5]. These compounds are promising drug candidates as they have been reported to have multiple anticancer effects in vivo and in vitro [5,26]. Among the components of Lithospermum erythrorhizon, b,b-Dimethylaerylshikonin has important anti-tumor effects compared to other active ingredients [27]. Regarding to anti-cancer activity, shikonin-rendered cell apoptosis through activation of caspase-dependent pathway was found in many malignant tumors. In this study, we investigated the effect of b,b-dimethylacrylshikonin on human gastric cancer SGC-7901 cells. Our results indicated that treatment of SGC-7901 cells with b,b-dimethylacrylshikonin showed a significant cytotoxic effect against SGC- The ratio of anti-and pro-apoptotic protein expression, such as Bcl-2/Bax, is crucial for the induction of apoptosis, and it decides the susceptibility of cells to undergo apoptosis [28]. Mitochondria play an important role in the signal transduction of apoptosis [29]. The translocalization of apoptotic proteins from the cytosol to the mitochondria leads to the release of cytochrome c and second mitochondria-derived activator of caspases by a decrease in mitochondrial membrane potential [30,31]. Shikonin has been reported to induce apoptosis via mitochondria pathway in HepG2 cells [32]. In the present study, we showed that b,b-dimethylacrylshikonin down-regulated the expression of XIAP, cIAP-2 and Bcl-2 and up-regulated the expression of Bak and Bax, and caused the loss of mitochondrial membrane potential and release of cytochrome c in SGC-7901 cells, consistent with mitochondria dependent apoptosis. The observation of b,b-dimethylacrylshikonin mediated activation of caspase-9, caspase-3, and subsequent cleavage of PARP, as well as the result that the pan-caspase inhibitor Z-VAD-FMK reduced b,b-dimethylacrylshikonin-induced apoptosis in SGC-7901 cells, suggesting that mitochondrial-mediated caspase cascade pathway plays a key role in b,bdimethylacrylshikonin-induced apoptosis. Taken together, our results indicated that b,b-dimethylacrylshikonin regulates expression levels of apoptosis-related proteins, causes cytochrome c release and trigs caspase-dependent cell apoptotic death.
Mitogen-activated protein kinases (MAPK) regulate diverse cellular programs including embryogenesis, proliferation, differentiation and apoptosis [33]. The MAPK family proteins are composed of three protein kinases: ERK1/2(extracellular signalregulated kinases 1 and 2), JNK (c-Jun N-terminal kinase) and p38 [34,35]. In general, transient ERK activation leads to cell proliferation, but sustained activation of ERK is assoctiated with differentiation [36]. Emerging evidence suggests that the activation of ERK contributes to apoptosis. Jeong et al. showed that kaempferol caused cancer cell to undergo apoptosis through an ERK-dependent pathway [21,37,38]. Recently, it has been reported that icaritin induces growth inhibition and apoptosis in human PSMCs via ERK signaling pathway [39]. Li et al. demonstrated that activation of RAF/MEK/ERK signaling pathway plays a critical role in calcium-induced apoptosis of lens epithelial cells [40]. Here we also found that the sustained activation of ERK is involved in b,b-dimethylacrylshikonininduced growth inhibition and apoptosis in SGC-7901 cells. U0126, a specific inhibitor of MEK (the MAPK/ERK kinase), effectively blocked b,b-dimethylacrylshikonin-induced ERK activation and attenuated b,b-dimethylacrylshikonin-induced apoptosis, suggesting the pro-apoptotic effects of b,b-dimethylacrylshikonin in SGC-7901 cells are mediated by the sustained activation of the ERK1/2 signaling pathway.
In conclusion, we found that b,b-dimethylacrylshikonin inhibited cell growth and induced apoptosis in SGC-7901 cells. We also studied the underlying mechanisms involved in b,b-dimethylacrylshikonin-induced apoptosis. Our results indicated that b,b- dimethylacrylshikonin-induced apoptosis involves in the reduction of XIAP, cIAP-2, and Bcl-2 protein expression and induction of Bak and Bax protein expression, and caused the loss of mitochondrial membrane potential and release of cytochrome c in SGC-7901 cells. Our results also demonstrated that b,bdimethylacrylshikonin induced apoptosis involves the activation of caspase-3, caspase-8 and caspase-9 and the cleavage of PARP. Besides, ERK signaling pathway also participated in b,bdimethylacrylshikonin-induced apoptosis. Our results indicated that b,b-dimethylacrylshikonin could be developed as a potential anticancer agent against human gastric cancer.