Background
Gastric cancer is the fourth common malignancy in males and the fifth in females. It accounts for about 9% of worldwide cancer deaths in 2012 [
1]. Most newly diagnosed cases present with locally advanced or metastatic gastric cancers [
2,
3] Still those patients with operable gastric adenocarcinoma would have a high risk of recurrence after tumor complete resection despite improvements in the surgical treatment [
4]. Chemotherapy has long been considered as a sophisticated treatment of locally advanced and metastatic gastric cancers [
5,
6]. Doxorubicin (DOX), an anthracycline-based anti-tumor drug combined with other chemotherapeutic agents including fluorouracil and mitomycin, was first introduced as a gold therapy regimen for advanced gastric cancer patients in 1980 [
7]. However, some clinical trials reported traditional DOX-based regimens failed to prolong survival and brought about some adverse effects for gastric cancer patients [
8‐
11]. Recent in vitro study indicated that the frequent acquisition of DOX resistance and the potential induction of epithelial–mesenchymal transition (EMT) might be crucial in the limitation of DOX treatment in gastric cancer [
12]. To find out a novel solution to alleviate the adverse effects in an all-round way, we endeavor to discover a chemo-sensitizer and side-effects attenuator for DOX in gastric cancer chemotherapy as well as its molecular mechanism.
A number of phytochemicals available exhibit chemo-preventive effect and sensitize cancer cells toward DOX [
13]. Among various phytochemicals, resveratrol (RES), a self-protective polyphenolic phytoalexin derived from plants, has attracted increasing attention due to its multiple health benefits [
14]. In addition to the pharmacological effects of RES on the aging process, diabetes, neurological dysfunction, cardiovascular system and inflammation, its potent anti-tumor activity were closely observed in studies including breast cancer, lung cancer, prostate cancer, hepatocaracinoma, colorectal cancer and so on [
14‐
16]. The combined treatment of DOX and RES achieves a synergistic effect and reverses multidrug-resistance in breast cancer cells [
17] and acute myeloid cells [
18]. As DOX is one of the key chemotherapy agents in gastric cancer treatment, the combination of DOX and RES turns out to be a potentially promising chemotherapy regimen for gastric cancer and the relevant mechanism remains to be explored.
In this study, DOX-resistant human gastric cancer cell line (SGC7901/DOX) was established and demonstrated aggressive migratory capabilities. Then RES was introduced and it successfully alleviated cell migration and chemo-sensitized DOX through reversing EMT process and regulating PTEN and Akt/mTOR pathway. Our work sheds new light on RES as a potential adjuvant to DOX in gastric cancer treatment to solve drug resistance and tumor metastasis. And the relevant mechanism study and prospective clinical trials are expected.
Methods
Cell culture
Human gastric cancer cells SGC7901 and MGC803 were obtained from American Type Culture Collection Cell Biology Collection (ATCC, Manassas, VA, USA) and maintained in Department of Pathology, Southern Medical University (Guangzhou, China). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator containing 5% CO2.
Establishment of DOX-resistant gastric cancer cell line
The DOX concentration gradient progressive increase induction method was used to develop DOX-resistant phenotype SGC7901 cell line. SGC7901 cells at a concentration of 1 × 105/ml were inoculated in DOX-free culture medium for 24 h until they got in the logarithmic phase. Then the culture medium was replaced with that containing low concentration (0.125 mg/L) DOX (Nanfang Hospital, Guangzhou, China, dissolved in RPMI 1640 medium) for 48 h. Then the culture medium containing drugs and dead cells was discarded. Cells were collected and re-inoculated in DOX-free culture medium to get recovered before next DOX treatment. After cells got adjusted to present DOX treatment, we increased the concentration of DOX and repeated the DOX and DOX-free treatment in turn until cells survived well and developed resistance to 0.75 mg/L DOX treatment. We referred to this DOX-resistant SGC7901 cell line as SGC7901/DOX. Both DOX- sensitive SGC7901 and DOX-resistant SGC7901/DOX were cultured under the same conditions and in drug-free medium for 2 days before commencing the experiments.
CCK-8 assay
Cell survival rates were estimated by the CCK-8 assay (keyGEN biotech, Jiangsu, China). Approximately 104 cells were seeded in 96-well plates with 100 μl medium each well. After 24 h cultivation, different doses of DOX or/and RES were added respectively. Each well was incubated with 10 μg CCK-8 solution for 2 h away from light before measuring the absorbance at 450 nm by PerkinElmer’s EnSpire Multilabel Plate Reader.
Cell scratch test
When the cells seeded in 6-well plate reached a confluent state, a single scratch was made using a sterile 10 μl pipette tip. The cells were then incubated with FBS-free culture medium (to exclude the potential effects of FBS on cell migration) alone or containing different concentrations of DOX or RES (SinaBestBio, Shanghai, China, dissolved in 10% DMSO (DMSO:RPMI 1640 medium = 1:9)). Images of the scratches were captured at 0, 24 and 48 h with Olympus IX71 inverted microscope at 100× magnification. The width of the scratch was analyzed using the Olympus CellSens Dimension software.
Transwell migration assay
Transwell migration assay was performed using transwell inserts (BD bioscience, SanJose, CA) with a filter of 8 μm pore. Cells were left untreated or treated with DOX, RES or both for 48 h. 8 × 104 cells in serum-free medium were seeded into the upper chamber of the insert and complete medium was added to the lower chamber. After 36 h incubation, the cells were fixed with methanol and stained with Giemsa. Then cells on the top surface of the membrane were wiped off, and cells on the lower surface were examined with Olympus DP72 microscope at 100× magnification. 4 random fields were photographed for counting purposes and the average number of migrated cells was used as a measure of migration capacity.
Cells were left untreated or treated with DOX, RES or both for 48 h. Then cells were trypsinized and dispensed into individual wells of six-well tissue culture dishes with a density of 300 cells per well. Following another 14 days in drug-free culture, cells were fixed and stained with Giemsa to visualize colonies. Experiments were performed in triplicate.
Apoptosis analysis
To detect cell apoptosis, cells were incubated with DOX or RES at different concentrations for 24 h. About 1 × 105 cells were harvested by EDTA-free trypsinization and washed twice with cold PBS. After being resuspended in 500 μL binding buffer, cells were stained with 5 μL Annexin-V-FITC and 5 μL PI (Keygen bioTECH, Jiangsu, China) in dark condition at room temperature for 15 min. Lastly, cell apoptosis was measured by FACSAria flow cytometer (BD, USA).
Immunofluorescence
The cells seeded in co focal dishes were fixed with 4% paraformaldehyde and then kept stable in 0.2% Triton for 10 min to rupture the cell membranes. Following three PBS washing, non-specific antigen-binding sites were blocked by 2% BSA for 30 min. The cells were then incubated with anti- Vimentin, E-cadherin (Abcam, 1:200); anti-β-catenin (Proteintech, 1:50) overnight at 4 °C. After washing, cells were incubated with anti-rabbit antibody for 60 min and the nuclei were stained with DAPI for 2 min, which was washed with PBS later. Cells were kept from light before observed with a fluorescence microscope.
Western blot analysis
Cells to be tested were homogenized in Lysis Buffer (containing 0.1% protease inhibitor, 0.5% 100 mM PMSF and 1% phosphatase inhibitor) and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and the total protein content was determined by BCA assay. Equal proteins (40 μg/lane) were loaded on SDS-PAGE gels and then transferred to 0.22 μm PVDF membranes. Membranes were blocked with 5% fat-free milk (5% skimmed milk powder in PBST, 0.1% Tween in PBS) and incubated with primary antibodies overnight at 4 °C. After being washed by PBST for 5 × 5 min, membranes were incubated with secondary antibodies for 1 h at room temperature. Following washing, target bands were visualized using Tanon-5200 Image Analyzer.
Primary antibodies used included rabbit monoclonal antibodies anti- PTEN, Akt, phosphor-Akt, mTOR, phosphor-mTOR, TSC1, TSC2, p70S6K, β-catenin, cleaved caspase-3 and caspase-9 (1:500, Cell Signaling Technology, Danvers, MA); rabbit monoclonal antibodies anti- Vimentin, E-cadherin (1:500, Abcam, Cambridge, UK); mouse monoclonal antibody anti-GAPDH (1:500, Proteintech, Chicago, IL). Protein bands were quantified using the Tanon-5200 Image Analyzer. All bands were normalized to GAPDH and the fold changes were calculated through relative quantification to the control group.
Nude mice xenograft model
Nude mice were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed under controlled temperature (28 °C) and pathogen-free conditions at Guangdong Provincial Key Laboratory of Molecular Tumor Pathology. All procedures were performed aseptically in accordance with the institutional guidelines of Guangdong Province and approved by the Experimental Animal Ethics Committee of Southern Medical University. 1 × 107 SGC7901/DOX cells were injected subcutaneously into the left inguinal region of each nude mouse when the mice got five weeks old. Two weeks later, mice were randomly divided into four groups (n = 5, 3 males, 2 females): Group 1 Control; Group 2 DOX (3 mg/kg); Group 3 RES (50 mg/kg); Group 4DOX (3 mg/kg) and RES (50 mg/kg). DOX was diluted in sodium chloride injection and given intraperitoneally (i.p.) while RES was dissolved in 10% DMSO and administered by gavage. Both drugs were given once a week for 4 consecutive weeks. The tumor volume (V) was determined by measuring the length (a) and width (b) with calipers every week and calculated using the formula: V (mm3) =1/2*ab2. At the end of the treatment period, mice were sacrificed, and tumors were collected for immunochemical studies.
Immunohistochemistry
The tumor tissue was fixed with 4% formalin and paraffin embedded before cutting it into 3 μm thick sections. And the sections were routinely deparaffinized in xylene and rehydrated in an alcohol gradient. Next, tissue antigen was retrieved by heating in sodium citrate (pH 6.0) or Tris-EDTA solution (pH 8.0) for 10 min. Sections were then blocked with 3% H2O2 before incubation with anti- caspase-3 (Cell Signaling Technology, 1:200); anti- ki67 (Cell Signaling Technology, 1:400); anti- Vimentin (Abcam, 1:500); anti-PTEN (Proteintech, 1:200) at 4 °C overnight. After three washes in PBS, the sections were incubated with secondary antibody (ZSGB-BIO, Beijing, China) for 30 min at room temperature. DAB kit (ZSGB-BIO, Beijing, China) was sequentially applied to stain the slides. Following counter-staining in hematoxylin and mounting with neutral balsam, the tissue sections were observed using Olympus BX-UCB light-field microscope. 5 random fields at × 400 magnification were captured for intensity analysis.
We used Image-Pro Plus software for semi-quantitative Image analysis. Firstly, we opened the image in the software and calibrated the optical density. In Measure menu we clicked Count/Size and chose Manual to enter into Segmentation dialogue box where the area of interest was set through: hue, 0 ~ 30; saturation, 0 ~ 255; intensity, 0 ~ 255, then the image was converted to gray scale image. Subsequently, we returned to Count/Size window, clicked Count and the values were measured. The parameters included area sum and IOD. The mean optical density was calculated by IOD/area and designated as representative staining intensity [
19,
20].
Statistical analysis
Data were expressed as means ± SD of at least three independent experiments. Statistical evaluation of the data was performed by using the unpaired Student’s t-test and ANOVA followed by a post-hoc test. Differences were considered to be statistically significant for p < 0.05. Statistical analysis was performed by SPSS 19.0 software.
Discussion
Chemotherapy, which provides palliation of symptoms and improves survival and life quality, is the most effective treatment for patients with inoperable cancers. However, conventional DOX-based chemotherapy regimen has been criticized for a series of negative effects, including the development of drug resistance and the occurrence of Epithelial–mesenchymal transition (EMT) [
21,
22].
EMT not only enhances the metastatic potentials of cancer but also participates in the development of chemo-resistance [
23,
24]. EMT is the physiological or pathological conversion of epithelial cells to mesenchymal cells, in which cells undergo phenotypic changes including the loss of cell polarity and cell-cell adhesion, the acquisition of migratory and invasive properties, which are highly responsible for carcinoma progression. The EMT-induced stemness endows cancer cells with the ability to overexpress chemo-resistance related genes, leading to multiple drug resistance in cancer treatment [
25]. A previous study detected DOX-induced EMT in BGC823 gastric cancer cells. Inhibition of β-catenin signaling could suppress DOX-induced EMT and cell migration [
22]. Suppression of EMT through selective inhibition of β-catenin signaling could restore sensitivity to HER-2 targeted lapatinib in HER-2 positive gastric cancer cells SNU216 cells [
26]. Very recently, an EMT lineage-tracing system was established to monitor reversible and transient EMT process in mice. Upon treatment with cancer chemotherapy drug cyclophosphamide, EMT cells were detected in the primary tumor and showed chemo-resistance owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Theses EMT cells also contributed to recurrent lung metastasis formation after chemotherapy. These data suggested that EMT plays an important role in cancer drug resistance and contributes to metastasis after chemotherapy treatment [
27]. In our study, we generated SGC7901/DOX cell line by long-term and incremental DOX treatment, which was characterized by the acquisition of drug resistance and enhancive migration (Fig.
2). And meanwhile, SGC7901/DOX cells displayed an apparent EMT potential for they were transformed into spindle-like shape, and expressed high level of mesenchymal cell markers including β-catenin and vimentin while losing epithelial cell adhesion molecule such as E-cadherin (Fig.
3).
EMT-mediated therapeutic resistance in solid tumors is regulated by many canonical signaling pathways, among which PI3K/Akt is of high interest. [
28,
29] PI3K phosphorylates PIP2 into PIP3, which then phosphorylates Akt in turn. Akt gets activated by phosphorylation of its Ser473 residue and stimulates the mTOR complex 1 (mTORC1) by phosphorylating tuberous sclerosis complex2 (TSC2) and subsequently inhibiting TSC1/2 complex formation, which is a negative regulator of mTORC1. Further, the mTORC1 complex acts on RHEB (Ras homolog enriched in brain) to phosphorylate mTOR at Ser2448 and thus resulting in mTOR activation. Then mTOR regulates protein translation and cell growth by phosphorylating ribosomal S6 kinase (p70S6K) [
30]. It has been reported that mTOR inhibitors decrease p70S6K and reverse resistance to DOX [
31]. Another important substrate of Akt that initiates the mitochondrial apoptotic pathway is caspase-9, which is inactivated by Akt through the phosphorylation at Ser196 [
32]. Caspase-9 inactivation then leads to the downregulation of caspase-3 and suppression of caspase-dependent apoptosis [
33]. As other study shows, Akt activation induces cancer cell invasiveness partially through interaction with vimentin. The binding of Akt to vimentin enhances the ability of vimentin to induce motility and invasion while protecting vimentin from caspase-induced proteolysis [
34]. Vimentin promotes cell invasiveness via the regulation of E-cadherin/β-catenin complex. [
35] Compared with E-cadherin and β-catenin, vimentin seemed to be a more specific and significant determinant for cancer metastasis [
36,
37]. Moreover, Akt triggers DOX-induced EMT through Akt/GSK-3β/β-catenin pathway. Nuclear translocation of β-catenin not only regulates EMT-associated proteins, which are responsible for cell migration, but also induces transcriptional upregulation of ZEB1, which in turn regulated DNA damage repair and DOX-resistance [
38]. Recently, the promotion of Akt phosphorylation could facilitate EMT, resulting in tumor formation and invasion in AGS and MKN45 gastric cancer cell lines [
39]. Given that Akt was activated in SGC7901/DOX cells, we are convinced that the upregulation of Akt signaling pathway best illustrates DOX-induced EMT, cell migration and drug resistance mentioned above. Even though activation of Akt signaling pathway was maintained by DOX treatment in SGC7901/DOX cells, caspase-3 mediated apoptosis was promoted by DOX in spite of caspase-9 inactivation by Akt. Therefore, DOX could induce SGC7901/DOX cell apoptosis in an Akt-independent way.
To alleviate the adverse effects of DOX, we took note of RES, a well-known phytochemical with diverse health benefits, as a possible supplement for DOX treatment. RES demonstrated its valid potentials as a chemosensitizer for DOX in breast cancer cells [
40]. In gastric cancer MGC803 cells, RES was able to induce cell cycle arrest by targeting PTEN. As a negative regulator of PI3K/Akt pathway which dephosphorylates PIP3, PTEN has been reported to reverse EMT-mediated drug resistance [
41]. Consistent with our data, an early study revealed RES is able to suppress cell invasion and the expression of Snail and N-cadherin and increase that of E-cadherin, which suggested that RES has an EMT-inhibitory effect in SGC7901 cells [
42]. We discovered that PTEN expression was enhanced by RES dominantly and dose-dependently in DOX-resistant SGC7901/DOX gastric cancer cells. Consequently, PI3K/Akt pathway was down-regulated, which activated caspase-3 dependent apoptosis, suppressed colony formation, and more importantly, inhibited EMT, leading to the suppression of cell migration and the reverse of DOX-resistance of SGC7901/DOX cells. Similar effects were also confirmed in SGC7901/DOX in vivo xenograpft tumor model.