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Usnic Acid Induces Cycle Arrest, Apoptosis, and Autophagy in Gastric Cancer Cells In Vitro and In Vivo

Background Usnic acid (UA), a secondary metabolite, is mainly derived from certain lichen species. Growing evidence suggests that UA has antitumor, anti-oxidative, anti-inflammatory, and other activities in a variety of cancer cells. However, the antitumor effect of UA in gastric cancer cells (GC) is unclear. The aim of this investigation was to assess the antitumor effect of UA in GC cells in vitro and in vivo, and to explore the underlying mechanisms. Material/Methods Cell proliferation was measured by CCK8 assay, the arrest of cell cycle was assessed by flow cytometry, and cellular apoptosis was observed via Hoechst 33258 staining assay. Expression levels of apoptosis-related proteins (activated caspase-3 and PARP, Bax, Bcl2) and autophagy-associated proteins (LC3-II and p62) were verified through Western blot analysis. H&E staining and immunohistochemistry were carried out in the subcutaneously implanted BGC823 tumor model in a nude mouse experiment. Results In vitro, we demonstrated that UA was significantly effective in inducing morphological changes, inhibiting the cell proliferation dose- and time-dependently, arresting the cell cycle phase, promoting cancer cellular apoptosis, and inducing autophagy activity. In vivo, compared to mice treated with 5-FU alone, UA treatment was significantly more effective in suppressing the tumor growth without affecting body weight, and in regulating the amount of Bax and Bcl2 in tumor tissues. Conclusions UA induces cell cycle arrest and autophagy and exerts anti-proliferative and apoptotic effects by modulating expression of apoptosis-related proteins in stomach neoplasm cells, and has a better antitumor effect compared to 5-Fu in the xenograft model.


Background
According to the Global Cancer Statistics of 2012, Gastric cancer is the fourth and the fifth most frequently diagnosed cancer and the third and the fifth leading cause of cancer-related death in men and women, respectively, worldwide [1]. In recent years, stomach cancer incidence has been declining in areas with historically high rates, but incidence rates are still high in China [1]. Surgery, as a primary treatment of choice for patients with GC, often is ineffective because many GC patients already have locally advanced or metastatic cancer disease at primary diagnosis, as well as due to recurrence after complete surgical resection, resulting in high GC mortality rates worldwide [2,3]. Chemotherapy, a conventional treatment strategy for metastatic GC, includes fluoropyrimidines, administered alone or in combination, such as with 5-fluorouracil (5-FU), capecitabine, and S-1, playing positive roles in inhibiting tumor growth [4]. Although 5-FU-based therapy is widely used in the standard first-line treatment of malignant GC, unexpected gastrointestinal toxicity and drug resistance greatly limit its clinical application [5,6]. Biomarkers of gastric cancer have also been studied, but mechanism still needs further exploration and confirmatory research [7,8]. In brief, GC still has a poor prognosis, with a low 5-year survival rate for patients diagnosed at advanced stages of GC [9,10]. Therefore, it is urgent to design new GC treatments that enhance therapeutic effects and reduce toxicity in the treatment of GC.
The cumulative data show that cancer cell death involves induction of cell cycle arrest, apoptosis, and autophagy, suggesting a possible strategy to deal with gastric cancer [11,12]. At present, investigation of cell cycle has been widely performed and the arrest of cell cycle phase has been shown to be very effective in the inhibition of cell viability [13,14]. Apoptosis (type-I PCD) is a form of programmed cell death (PCD) that can be activated through 2 dominating signaling pathways, consisting of the extrinsic (death receptor pathway) and intrinsic (mitochondrial pathway) pathways [12]. In addition, apoptosis pathways also involve the B cell lymphoma-2 (Bcl-2) family proteins, which are divided into 2 groups of pro-apoptotic proteins (Bax, and Bak and Bad) and a third group of antiapoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1) [15]. Apart from the pro-survival effect of autophagy, autophagy can also facilitate cell death (type-II PCD) in some cell types under certain intensities of stimulation [16].
However, it is still not well-established that UA induces cell cycle arrest, cell apoptosis, and autophagy in GC cells in vitro and in vivo. Therefore, in the present study, we investigated the functional roles of UA with associated molecular alterations in vitro and in an in vivo xenograft tumor model.

Material and Methods
Chemicals and UA treatment, cell lines, and cell culture (+)-usnic acid powder (UA, #329967, HPLC, purity ³98%, Figure 1A) and 5-fluorouracil (5-FU, #F6627, HPLC, purity ³99%) were obtained from Sigma-Aldrich. We prepared a 50-mM stock solution of UA (in DMSO) and a 50-mM solution of 5-FU (in DMSO) kept in the dark at -20°C, and then freshly diluted at required concentrations in cell culture medium or phosphate-buffered saline (PBS, #KGM20012) prior to use in each experiment. We used a 0.1% final concentration of DMSO as the control. In the cell viability assay, UA was added to prepared concentrations of 31. 25   To assess the viability of the human GC cells treated with UA, the Cell Counting Kit-8 assay was performed according to the manufacturer's protocols. Briefly, BGC823 and SGC7901 cells were seeded into 96-well plates (6000-8000 cells/well) with a total volume of 100 µl medium per well, and allowed to attach for 24 h. Then, the cells were treated with a series of corresponding concentrations of UA (0-1000 µM) for 24 h and 48 h. At the end of incubation, the medium was removed, and the cells were treated with 10% CCK-8 (Dojindo Laboratories, Kumamoto, Japan) in 100 μl RPMI-1640 medium without FBS for 2 h in the dark at 37°C. We measured the absorbance of each well at 450 nm by using a microplate reader (ELX808; Bio Tek, Winooski, VT, USA) and the half-maximal inhibitory concentration (IC50) values were calculated using probit analysis of SPSS version 19.0. Cell viability was calculated according to the following formula: the viability ratio (%) =[(O1-O3)/ (O2-O3)]×100, where, O1 is the OD value of drug experimental group, O2 is the OD value of blank control group (0 µM of UA), and O3 is the OD value of the RPMI1640 medium without cells.

Cell morphology assay (Inverted Optical Microscopy)
We further observed the changes in cell behavior of UA-treated BGC823 and SGC7901 cells. Briefly, cells were plated in 6-well plates (5×10 5 cells per well). At 40-60% confluence, culture medium was replaced with fresh medium with various concentrations of UA, and then cells were incubated for a further 24 h. Cells morphological changes were observed by use of an inverted microscope (Olympus Corporation, USA).

Cell cycle analysis by flow cytometry
Flow cytometry (BD FACS Calibur™; Becton-Dickinson, Franklin Lakes, NJ, USA) was used to analyze the cell cycle distributions using the Cell Cycle Staining Kit (PI/RNase Staining Buffer #550825, BD Pharmingen, USA) according to the manufacturers' instructions. In brief, human GC cells were seeded in 6-well plates at a density of 5.0×10 5 cells/well. After 24 h, the medium was removed and replaced with fresh medium containing a graded concentration of UA for another 24 h. The cells were then harvested and cell suspensions were pelleted and washed by centrifugation at 1000 rpm at 4°C. Cells were then fixed in cold 70% ethanol at -20°C overnight. After that, ethanol-fixed cells were centrifuged at 1000 rpm at room temperature and washed twice with cold PBS and FACS buffer. Then, single-cell suspensions at a density of 1×10 6 of BGC823 or SGC7901 cells were resuspended in PI/RNase Staining Buffer and incubated for 15 min in the dark at room temperature and transferred to flow cytometry tubes for cell cycle analysis at slow flow rate and then analyzed in the ModFit LT5.0 program (http://www. vsh.com/products/mflt/mfTrialVersions.asp).

Cell cellular apoptosis assay by Hoechst staining
Hoechst33258 staining assay was used to evaluate cellular apoptosis. The procedure for Hoechst staining was carried out using Hoechst33258 (#C0003, Beyotime, Shanghai, China) kits according to the manufacturer's instructions. Briefly, cells in 6-well plates were exposed to UA for 24 h, then the GC cells were fixed for 10 min in 4% paraformaldehyde, washed in cold PBS, and incubated at room temperature for 5 min with Hoechst33258 in the dark. Stained cells were washed twice with PBS and then observed with a fluorescence microscope (Leica, Wetzlar, Germany) under UV excitation.

Western blotting
For Western blot analysis, cells were seeded in 6-well plates at a density of 1×10 6 [27,28] and our pilot trial. In the process of drug administration, the weights of tumor-bearing mice and tumor diameters were recorded once every 2 days. One hour after the last treatment, the mice were killed humanely to collect the tumors. Tumor volumes were determined using the following formula: V=0.5×L×W 2 , where, V is the volume, L is the length, and W is the width.

Histological assessment
The tumor tissues were cut and fixed using 4% paraformaldehyde for 24 h at room temperature and then dehydrated and embedded in paraffin. After deparaffination and rehydration, 2-μm-thick tissues were mounted on polylysine-coated slides for H&E staining. Images of H&E staining were obtained using a microscope. Using immunohistochemical method, the presence of Bax (#GB11007, Servicebio, 1: 300) and Bcl-2 (#GB11008, Servicebio, 1: 300) in the paraffin-embedded tumor tissues were assayed. Staining was performed as defined in the manufacturer's staining protocol. Samples were randomly selected from the tumor tissues. Immunostainings were performed with known positive and negative tumor controls. Immunohistochemical staining slides were scanned by a highspeed digital slide microscopy scanners-Panoramic MIDI with a 20× microscope objective (3DHISTECH, Hungary). Computer analysis of staining intensity of IHC reaction image of all areas was performed and the number of stained pixels/area was quantified using the Densito Quant module (3DHISTECH, Hungary). An intensity score of 0 to 3 was assigned to the in- The maximum H-score could be 300, corresponding to 100% of cells with strong intensity.

Statistical analysis
Statistical analyses were performed using software SPSS version 19.0 (IBM, Armonk, NY, USA) and software Graph-Pad Prism version 7.0 (Windows, Graph-Pad Software, San Diego, California, USA, www.graphpad.com.). All in vitro experiments were performed independently at least in triplicate. Statistical significant differences were determined by a two-tailed t test when comparing 2 groups. P<0.05 was considered as statistically significant. Data are expressed as mean ± standard deviation (SD).

UA induces the inhibition of cell proliferation.
In vitro antitumor capabilities of UA were evaluated using the CCK8 assays. Serially diluted UA solutions were added to BGC823 and SGC7901 cells and incubated for 24 h and 48 h. As shown in Figure 1B, 1C, cell viability decreased in a concentration-dependent and time-dependent manner when treated with UA. The IC50 (24h) of UA in BGC823 cells was 236.55±11.12 µM, which was significantly lower (P=0) than the IC50 (24 h, IC50=618.82×1.77 µM) of SGC7901 cells, suggesting that BGC823 cells were more sensitive than SGC7901 cells to UA.

UA induces cell morphological change.
By observation under an inverted microscope, we found that GC cells growth was significantly decreased at the experimental concentration of UA. After treatment with UA for 24 h, a reduction in cell volume, shrunken cytoplasm, extensive vacuolization, and some cellular damage were observed, including most cells detached from culture surfaces, in a dosedependent manner ( Figure 1D, black arrows).

UA induces cell cycle arrest of G0/G1 or G2/M phase
To explore whether UA mediated the inhibition of GC cells growth via cell cycle arresting mechanisms, we analyzed cell cycle distribution subsequent to the cells being treated with indicated concentrations of UA for 24 h, using flow cytometry (PI staining). As shown in Figure 2, we found that UA treatment resulted in an accumulation of BGC823 cells in the G0/G1 phase from 54.4±2.7% to 66.9±2.6% (UA: 400 µM, P=0.005) and a significant decrease of BGC823 cells in the S phase (P=0.043) from 39.7±4.7% to 28.7±4.5% as compared with the 0.1% DMSO control, and the effect was not dose-dependent (Figure 2A, 2C). UA exposure also induced an increase in G2/M phase cell population (from 4.7±3.9% to 16.7±4.4%, p=0.024, UA: 1200 µM) and a decrease in the S phase cell population (from 48.4±4.3% to 34.9±5.8%, p=0.031) in SGC-7901, and the effect was not dose-dependent ( Figure 2B, 2D), suggesting that UA inhibited the growth of GC cells, partly resulting from inducing cell cycle arrest.

UA induces cell apoptosis and autophagy.
Chromatin condensation, a typical morphological change characteristic of apoptosis, was observed after Hoechst 33258        Figure 3A, yellow arrows). The possible signaling pathways through which UA induces cell apoptosis in GC cells were investigated. Western blot analysis revealed that the protein expression levels of Bax, activated caspase-3, and PARP were increased, but the level of Bcl-2 in GC cells was decreased, which led to cell apoptosis ( Figure 3B). Some compounds have been demonstrated to induce autophagic cell death [29]; therefore, we decided to test whether UA could also induce the expression of autophagy-associated protein in GC cells. The bands showed that the protein expression levels of LC3-II and p62 were increased and decreased, respectively, in BGC823 and SGC7901 cells ( Figure 3C).

UA induces the inhibition of tumor growth in BGC823bearing mice
To assess the antitumor effect of UA in vivo, a BGC823 subcutaneous graft model was established. The weight of mice and the volume of tumors were positively correlated with the severity of tumors. There was no conspicuous difference in the weight and the tumor size among different groups prior to the drug treatment (P>0.05). As shown in Figure 4, the control group of animals with transplanted BGC823 cancer cells showed a progressive increase in their tumor volumes ( Figure 4B) and tumor weight ( Figure 4C, 4D). Compared to the control group, the tumor sizes ( Figure 4B) and tumor weight ( Figure 4C, 4D) of tumor-bearing mice in the UA group were significantly decreased (p<0.05) without weight loss ( Figure 4A), while the decrease (P>0.05) in the 5-FU group was accompanied by weight loss (p<0.01) after the corresponding administrations. Moreover, UA administration was observed to exert a stronger antitumor effect on the tumor size and tumor weight compared to the 5-FU group (p<0.01). Therefore, UA treatment potentially attenuated side-effects of the agent and might be beneficial against GC cells.
UA induces the histopathological changes of tumor tissue.
As illustrated in Figure 4E-4I, the necrosis (yellow arrows) inside the tumor tissues (stained by H&E) showed the inhibitory efficacy of UA ( Figure 4E). In immunohistochemistry (IHC) analysis for cell apoptosis, Bax-positive tumor cells were significantly increased after treatment with UA as compared to mice in the control and 5-FU groups ( Figure 4F, 4H, p=0.001, p=0.002) while Bcl2-positive tumor cells were remarkably reduced after UA administration as compared to mice in the control and 5-FU groups ( Figure 4G, 4I, p=0.002, p=0.019). These results show that UA exerted better antitumor growth as compared with control and 5-FU groups.

Discussion
Recently, usnic acid has been reported to exert an antitumor effect against various cancer cells by inducing cell cycle arrest, cell apoptosis, and autophagy, leading to the inhibition of cell proliferation and cell death [22. However, research on UA is still insufficient in GC cells. Therefore, in the present study, we examined the antitumor effects of UA in GC cell lines, as well as the underlying mechanisms, both in vivo and in vitro.
The present results indicate that UA inhibited the GC cell growth in vitro in a dose-dependent and time-dependent manner, using CCK-8 growth assay ( Figure 1B, 1C). Uncontrolled proliferation resulting from abnormal activity of multifarious cell cycle proteins is a feature of cancer [30]. Data from cell cycle distribution assay by flow cytometry showed that UA induced G0/G1 or G2/M phase arrest in BGC-823 (Figure 2A, 2C) and SGC7901 ( Figure 2B, 2D) cells with no dose-dependent response, making GC cells undergo apoptotic progression. This finding is similar to previous studies in which UA was revealed to induce G0/ G1 phase arrest in human lung cancer [24] and G2/M phase arrest in human AGS cancer cells [25]. The Hoechst 33258 staining assay showed that treatment with UA also triggered cell apoptosis ( Figure 3A). In addition, apoptosis and autophagy induced by UA were validated by measuring the level of apoptosis -( Figure 3B) and autophagy -( Figure 3C) associated proteins by Western blot. All these findings are consistent with previous studies showing that UA induces cell cycle arrest, apoptosis, and autophagy in some cells, including breast cancer and lung cancer [23,26]. All results mentioned above indicated that UA can suppress the proliferation of human GC cells by inducing cycle phase arrest, cell apoptosis, and autophagy.
Apoptosis is a type of cancer cell death and is considered a crucial target for cancer therapy [31]. Typically, activation of the extrinsic (death receptor-mediated) and the intrinsic (mitochondrial-mediated) pathways participate in the induction of cellular apoptosis [32]. In both apoptotic pathways, caspases (cysteinyl proteases family) are the key components, cleaving target proteins. Procaspase-8 is activated by membrane-associated protein complexes (Fas-L/TNF) in the extrinsic pathway, while procaspase-9 is activated by mitochondria-associated protein in the intrinsic pathway. Then, procaspase-3 is activated at the convergent point of the 2 pathways and the target proteins (PARP proteins) of cleaved caspase-3 are activated afterwards [33]. The present results revealed that UA treatment led to an increase of cleaved PARP and activated caspase-3, confirming that UA can induce apoptosis via activation of a caspase cascade. In addition, the BCL-2 family also plays major roles in apoptosis, covering anti-apoptosis protein (BH1-4 domains) and pro-apoptosis protein (BH1-3 and BH3 domains) [34]. Specifically, the Bcl-2 subfamily (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1/Bfl-1) is associated with the inhibition of apoptosis, whereas the Bax subfamily (Bax, Bak, and Bok/Mtd) and the BH3-only subfamily (Bid, Bad, Bim/Bod, Bmf, Bik/Nbk, Blk, Noxa, Puma/Bbc3, and Hrk/DP5) promote apoptosis [35].
In the present study, UA treatment up-regulated the expression of pro-apoptotic protein Bax and down-regulated the expression of anti-apoptotic protein Bcl-2, suggesting the induction of apoptosis by UA in BGC-823 and SGC-7901 cells through regulating Bcl-2 family proteins. Previous studies revealed that UA induced apoptosis through the mitochondrial apoptotic pathways in cancer cells [26,27]. Autophagy is a low-level essential catabolic process of self-degradation and recycling of cellular constituents involving double-membraned vesicles and lysosomes [27]. This process can be accelerated in cancer cells under cellular stressors such as nutrient starvation, DNA damage, and organelle damage [36].
Although autophagy is considered a protective mechanism against ischemia in tumors, hyper-activation of autophagy can lead to cell death [37]. LC3 proteins, especially LC3-II, are recognized as the most common markers of autophagy, and P62 is incorporated in the completed autophagosomes and then degraded in autolysosomes [38,39]. The present study demonstrated that UA induced autophagy via the down-regulation of p62 and the up-regulation of LC3-II. A previous study showed that UA induces autophagic cell death in cells via inhibition of PI3K/AKT/mTOR [40].
The present study showed that UA suppressed the human GC cells (BGC823) tumor formation in the xenograft model in vivo (Figure 4). The mean tumor volume of UA-treated mice was smaller compared with controls ( Figure 4B-4D). UA treatment reduced tumor burden and increased the number of necrotic cells inside the tumors, compared with tumors from PBStreated mice ( Figure 4E). Staining of apoptosis-associated proteins ( Figure 4F-4I, Bax, Bcl2) confirmed the cells apoptosis by UA since the tumor cells in UA group had a significantly larger population of Bax and a smaller population of Bcl2positive cells as compared to the PBS group. A previous study showed that the growth of lung cancer tumors was inhibited in UA-pretreated cancer cell xenografts inhibited at the dose of 10 µM [25]. The results of the present study are in agreement with those of some previous studies, including studies on hepatoma cells [28], and further illustrates that UA may also be used in different cancers.

Conclusions
In conclusion, results revealed that UA can induce autophagy and inhibit the proliferation of human GC cells in vitro by inducing phase arrest and cell apoptosis. UA treatment also has a better antitumor effect than 5-Fu in vivo. All data from the present study suggest that UA is an attractive and promising candidate for developing antitumor drugs targeting GC.

Conflicts of interest
None.