Rosmarinic Acid Analogue-11 Induces Apoptosis of Human Gastric Cancer SGC-7901 Cells via the Epidermal Growth Factor Receptor (EGFR)/Akt/Nuclear Factor kappa B (NF-κB) Pathway

Background

Cancer is a major public health problem worldwide and the second leading cause of death in humans [1]. According to the latest statistics from the American Cancer Society, there will be 1.73 million cancer cases and more than 600 000 cancer deaths in the United States in 2018, among which there will be 26 240 new cases of gastric cancer and around 10 800 deaths arising from gastric cancer [2]. As such, gastric cancer should be considered a worldwide public health problem [3]. Surgery and chemotherapy are the main treatment methods for gastric cancer, and have been effective in reducing mortality, but the 5-year survival rate of gastric cancer patients is still relatively low [4]. Nowadays, various new natural compounds, such as podophyllin, paclitaxel, camptothecin, and vinblastine, have been isolated and used as effective drugs for cancer treatment, but their side effects are relatively large [5]. So, finding a new drug to treat stomach cancer is crucial [6]. Rosmarinic acid (RA) is a type of natural phenol carboxylic acid, which is a secondary metabolite commonly used in cooking herbs and commonly found in herbs such as rosemary, sage, thyme, and mint [7]. RA has a variety of promising biological effects, including anti-cancer [8], anti-microbial [9], anti-anaphylaxis, anti-inflammatory [10], and antioxidant [11]. For instance, Rocha et al. identified the anti-inflammatory potential of RA in rat models of local and systemic inflammation for the first time [10]. Han et al. found that RA might potentially be a therapeutic agent for suppressing the Warburg effect in gastric carcinoma [8]. Hence our research group synthesized a dozen RA analogues (RAAs). The preliminary experiment found that RAA-11 had the most prominent anti-cancer effect, with a molecular weight of 377.34. Its molecular structure formula and synthesis route are shown in Figure 1. Its MS and 1H-NMR are shown in Figures 2 and 3 respectively. Therefore, this study applied RAA-11 to intervene on human gastric cancer SGC-7901 cells to understand its effects on cell proliferation and apoptosis, and explore their apoptosis mechanism so as to provide fundamental theories for further research.

Material and Methods

MAIN REAGENTS AND PREPARATION:

RAA-11 was synthesized from the College of Pharmacy, Guangxi Medical University, with a purity of 98% or more. Brief synthetic steps are described as follows: 1.21 g methyl ester hydrochloride was dissolved in 5 mL DMF and stirred at 0–5°C for 10 minutes. Then the solution had added, drop wise, 0.75 g of 3, 4-difluorocinnamic acid dissolved in 10 mL dichloromethane solution as well as 2.12 g PyBOP and 1.63 mL triethanolamine. After reaction for 30 minutes, the mixture was stirred for 16 hours at room temperature and then extracted with ethyl acetate, then successively washed with 1 M hydrochloric acid solution, 10% sodium bicarbonate solution, water, and saturated sodium chloride solution. Then the solution was dried with anhydrous sodium sulphate to harvest a 1.32 g flavescent oily substance (RAA-11). RAA-11 was dissolved in DMSO to make into 50 mmol/L stock solution, and preserved at −20°C away from sunlight. During the experiment, RAA-11 stock solution was diluted to high, medium, and low concentration (40 μmol/L, 20 μmol/L, and 10 μmol/L respectively) with the Roswell Park Memorial Institute-1640 (RPMI-1640) Medium supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin. Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Becton, Dickinson and Company (New York, NY, USA). AxyPrep™ Multisource Total RNA Miniprep Kit was purchased from Axygen BioScience, Inc. (United City, CA, USA). RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) was purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). Epidermal growth factor receptor (EGFR) mRNA and GAPDH mRNA were purchased from GenScript Biotech Corp. Co., Ltd. (Nanjing, Jiangsu, China). EGFR, Akt, p-Akt, NF-κB, GAPDH, BAX, Bcl-2, and caspase-3 rabbit anti-human monoclonal antibodies were purchased from Wanleibio Co., Ltd. (Shenyang, Liaoning, China). Dylight 680 AffiniPure Goat Anti-Rabbit IgG (H+L) was purchased from EarthOx, LLC (San Francisco, CA, USA).

APPARATUS:

The inverted fluorescence microscope was purchased from Nikon Corporation (Tokyo, Japan). The BX53 positive fluorescence microscope was purchased from Olympus Corporation (Tokyo, Japan). ELx800 microplate reader was purchased from BioTek Inc. (VT, USA). Flow Cytometry was purchased from Becton, Dickinson and Company (New York, NY, USA). NanoDrop 2000 Spectrophotometer was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). StepOnePlus™ Real-Time PCR System was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). SDS-PAGE electrophoresis meter and Trans-Blot SD were purchased from Bio-Rad (Hercules, CA, USA). LI-COR Odyssey 2-tone infrared fluorescence imaging system was purchased from LI-COR Biosciences Inc. (Lincoln, NE, USA).

CELL CULTURE:

Human gastric mucosal epithelial cell lines GES-1 and human gastric cancer cell lines SGC-7901 were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in RPMI-1640 medium with 10% FBS and 100 μg/mL streptomycin, and 100 U/mL penicillin in the 37°C in a 5% CO₂ incubator. When the cells grew exponentially, they were digested with trypsin.

3-(4,5-DIMETHYL-2-THIAZOLYL)-2,5-DIPHENYL-2-H-TETRAZOLIUM BROMIDE (MTT) ASSAY:

The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide) assay was performed as described previously [12]. Human gastric mucosal epithelial GES-1 cells and human gastric cancer SGC-7901 cells were routinely cultured and digested at exponential phase. The cells were diluted into a concentration of 5×104 cells/mL by RPMI-1640 medium containing 10% FBS and inoculated into 96-well plates with 100 μL per well. The culture solution was discarded after the cells adhered to the wall. Then, 200 μL of RAA-11 with 0, 10, 20, 40, 80, and 160 μmol/L was added to each well, and 5 duplicate wells were set for each dose group. After the cells were cultured for 24 hours, 36 hours, and 48 hours, 20 μL of 5 mg/mL MTT was added per well and cultured for 4 hours in a 37°C incubator. Then each well had added 100 μL DMSO for 10 minutes. The optical density (OD) value of the each well was determined using the 570 nm microplate reader to calculate cell survival.

COLONY FORMATION ASSAY:

Colon formation assay was performed as described previously [13]. When the human gastric cancer SGC-7901 cells were digested at exponential phase, the cell suspension was diluted to 1×103 cells/mL. After manipulating them to single cell suspension, 500 μL cell suspension per well were placed in a 6-well plate with 1 mL RPMI-1640 medium containing 10% FBS. Then 2 mL of RAA-11 (0, 10, 20, and 40 μmol/L) were added to each well after the cells adhered to the wall, and the RPMI-1640 medium was replaced on alternate days to make the cells grow for 1 to 2 weeks (when the colony was visible to the naked eye). The cells were washed 2 times with phosphate buffer saline (PBS), and fixed in 4% paraformaldehyde fix solution for 15 minutes after air-dried at room temperature. Then the cells were dyed for 20 minutes with 1% crystal violet staining solution. Images were taken under the inverted fluorescence microscope to count the Colony Forming Unit (CFU).

HOECHST33258 STAIN ASSAY:

The Hoechst33258 stain assay was performed as described previously [14]. When the human gastric cancer SGC-7901 cells were digested at exponential phase, the cell suspension was diluted to 2×104 cells/mL. Then 2 mL of cell suspension were inoculated into a 6-well plate with embedded with aseptically treated cover glass. Then 2 mL of RAA-11 (0, 10, 20, and 40 μmol/L) were added to each well after the cells adhered to the wall. After 48 hours, the cells were washed with PBS twice, and fixed with 1 mL of 4% paraformaldehyde fix solution at room temperature for 20 minutes. Then, the cells were stained with 500 μL Hoechst33258 stain solution at room temperature away from light for 15 minutes. Finally, the cells were gently rinsed twice with PBS. The cover glass was placed on the clean microslides and sealed with a drop of Antifade Mounting Medium. The fluorescence intensity of the Hoechst33258 was observed using BX53 positive fluorescence microscopy.

FLOW CYTOMETRY:

Flow cytometry was performed as described previously [14]. When the human gastric cancer SGC-7901 cells were digested at exponential phase, the cell suspension was diluted to 1.5×105 cells/mL and 2 mL of the cell suspension were inoculated into a 6-well plate. Then 2 mL of RAA-11 (0, 10, 20, and 40 μmol/L) were added to each well after the cells adhered to the wall. After 48 hours, the supernatant and cells were centrifuged for 5 minutes at 1000 rpm. Then 1 mL iced PBS was added to resuspend the cells, and centrifuged for 5 minutes at 1000 rpm.200 μL 1×Binding Buffer was added to resuspend cells, followed by 5 μL propidium iodide (PI) and 5 μL Annexin V-FITC to incubate for 15 minutes at room temperature. Then 300 μL 1x Binding Buffer was added. The cell suspension was filtered through 300 mesh filter once and tested within 1 hour.

QUANTITATIVE REAL-TIME PCR:

Quantitative real-time PCR (qRT-PCR) was performed as described previously [15]. When the human gastric cancer SGC-7901 cells were digested at exponential phase, they were cultured in 25 cm2 culture bottles for 24 hours and treated with 3 mL of RAA-11 with final concentration at 0, 10, 20, and 40 μmol/L respectively. After 48 hours, the supernatant and cells were collected, and centrifuged for 5 minutes at 2000 rpm. Then 400 μL Buffer R-I was added to resuspend the cells and then transferred to a 1.5 mL centrifuge tube. 150 μL Buffer R-II was added in the 1.5 mL centrifuge tube, followed by centrifuge for 5 minutes at 12 000 rpm at 4°C. The supernatant fluid was transferred into a 1.5 mL centrifugal tube, and 250 μL of 2-propanol was added to the tube and the mixture was mixed evenly. The mixture was put in a 2 mL centrifuge tube and centrifuged for 1 minute at 6000 rpm at 4°C. The filtrate was then discarded. We added 500 μL Buffer W1A and centrifuged for 1 minute at 12 000 rpm. The filtrate was then discarded once more and 700 μL Buffer W2 was added and centrifuged for 1 minute at 12 000 rpm. The last process was repeated once more. The preparation tube was put into a new 1.5 mL centrifuge tube and 30 μL Buffer TE was added in the preparation tube. Finally, the cells were centrifuged for 1 minute at 12 000 rpm and eluted to get RNA. The NanoDrop 2000 Spectrophotometer was used to detect the RNA concentration, after which the RNA was put at −80°C for storage. Reverse transcription was performed on ice according to kit instruction, and the reaction system of each sample is shown in Table 1. The First Strand cDNA Synthesis was mixed gently and centrifuged briefly, then incubated for 60 minutes at 42°C and the reaction was terminated by heating at 70°C for 5 minutes. The cDNA can be stored at −20°C for less than 1 week.

Based on the gene sequences of human GAPDH and EGFR in the NCBI Gene Bank, the primers were designed and synthesized by GenScript Biotech Corp., Co., Ltd., with the sequences shown in Table 2.

PCR amplification was performed on ice according to kit instruction, and the reaction system of each sample is shown in Table 3.

The data was analyzed by a relatively quantitative analysis. The 2^−ΔΔCt values were calculated to compare the level of gene expression in various groups. All values were normalized against GAPDH values.

WESTERN BLOT:

Western blot was performed as previously described [12]. When the human gastric cancer SGC-7901 cells were digested at exponential phase, they were cultured in 25 cm2 culture bottles for 24 hours and treated with 3 mL of RAA-11 with final concentration at 0, 10, 20, and 40 μmol/L respectively. After 48 hours, the supernatant and cells were collected and washed with PBS 3 times. The cells were lysed on ice for 20 minutes with Lysis Buffer (RIPA: PMSF: Cocktail=100: 1: 1), centrifuged for 20 minutes at 12 000 rpm at 4°C. After which BCA Protein Assay was used to detect protein concentration, the supernatant was boiled with SDS-PAGE Sample Loading Buffer for 5 minutes. Then 40 μg samples of protein were separated by electrophoresis at 60V constant pressure. When the samples reached the separation gel, the voltage was changed to 120V until samples reached the bottom of the gel. Trans-Blot SD was used to transfer the protein onto the polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were washed 3 times with TBST and sealed with 5% BSA at room temperature for 30 minutes. Next, the PVDF membranes were washed 3 times with TBST and incubated with rabbit anti-human monoclonal antibodies overnight at 4°C and then incubated with Dylight 680 AffiniPure Goat Anti-Rabbit IgG (H+L) under the conditions of protection from light at room temperature for 1 hour. After the PVDF membranes were washed 3 times with TBST away from light, the films were scanned on Odyssey Infrared Imaging System. All values were normalized against GAPDH values.

STATISTICAL ANALYSIS:

SPSS20.0 statistics software (SPSS Inc., USA) was used to carry out the statistical analysis, measured values were expressed with mean ± standard deviation (χ̄±S), The t-test was used for the comparison between groups, single factor analysis of variance was used when there was comparison of more than 2 groups. Bilateral test was selected. P<0.05 value was considered as statistically significant and all techniques were replicated 3 times.

Results

EVALUATION OF SURVIVAL RATE BY USING MTT ASSAY:

As shown in Tables 4 and 5, the basal proliferation rate of SGC-7901 cells exhibited statistically significant differences compared with GES-1 cells. The inhibitory effect on the proliferation of SGC-7901 cells increased in a time- and concentration-dependent manner. Table 6 shows that the IC50 of RAA-11 at 24 hours, 36 hours, and 48 hours on GES-1 cells was 289.425±0.854 μmol/L, 220.430±2.300 μmol/L, and 189.521±1.002 μmol/L respectively, whereas the IC50 of RAA-11 at 24 hours, 36 hours, and 48 hours of SGC-7901 cells was 73.299±2.011 μmol/L, 50.426±1.458 μmol/L, and 37.684±0.579 μmol/L respectively.

EVALUATION OF CELL COLONY FORMATION ABILITY BY USING COLONY FORMATION ASSAY:

As shown in Figure 4, compared to the 0 μmol/L group (P<0.001), the colony inhibition rate of SGC-7901 cells increased in a concentration-dependent manner. The colony inhibition rates of RAA-11 at 0 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L were 12.68%, 56.48%, 75.64%, and 83.66% (colony inhibition rate (%)=(CFUcontrol group–CFUdose group)/CFUcontrol group×100%).

EVALUATION OF MORPHOLOGICAL CHANGES OF APOPTOSIS BY USING HOECHST33258 STAIN ASSAY:

Hoechst33258 assay combines with nuclear condensation and fragmentation in the apoptotic cells, showing a strongly fluorescent reaction. The viable non-apoptotic cells do not undergo pyknotic nucleus or rhexes but show relatively light fluorescence. As shown in Figure 5, compared to the 0 μmol/L group (P<0.001), the SGC-7901 cells showed obvious apoptosis after treatment with RAA-11 for 48 hours, and the cell apoptotic rate (cell apoptotic rate(%)=apoptotic cellular score/total cellular score×100%) increased significantly in a dose-dependent manner.

EVALUATION OF APOPTOSIS RATE BY USING FLOW CYTOMETRY:

The effects of RAA-11 on apoptosis include early apoptosis (Annexin V+/PI−) and late apoptosis (Annexin V+/PI+). As shown in Figure 6, the apoptotic rate was 4.09% (0 μmol/L), 39.77% (10 μmol/L), 42.72% (20 μmol/L), and 51.12% (40 μmol/L), respectively. The data exhibited RAA-11 could induce apoptosis of SGC-7901 cells in a dose-dependent manner (P<0.001).

EVALUATION OF EXPRESSION LEVEL OF EGFR MRNA BY USING QRT-PCR:

As shown in Figure 7, the expression level of EGFR mRNA was significantly downregulated compared to the 0 μmol/L group (P<0.001), indicating that RAA-11 can downregulate the mRNA expression level of the EGFR gene in SGC-7901 cells.

EVALUATION OF EXPRESSION LEVEL OF RELATIVE PROTEINS BY USING WESTERN BLOT:

As shown in Figures 8 and 9, the expression of inhibitory apoptotic protein Bcl-2 was decreased and the expression of pro-apoptotic protein BAX and caspase-3 was increased when compared with the 0 μmol/L group (P<0.001). The expression of pathway proteins EGFR, Akt, p-Akt, and NF-κB was significantly downregulated (P<0.001), suggesting that RAA-11 may induce apoptosis of SGC-7901 cells by inhibiting the EGFR/Akt/NF-κB pathway.

Discussion

Rosemarinic acid (RA) is a kind of polyphenols hydroxyl compound [16]. It contains a molecule of caffeic acid and a molecule of 3,4-dihydroxy phenyl lactic acid [17]. In recent years, more and more attention has been paid to rosemary, with numerous reports on the chemical composition and pharmacological effects of rosemary [18]. Nowadays, the treatment of malignant tumors has become a major difficulty that we urgently need to overcome. RA is rich in a variety of plants, and it can play an anti-tumor role through a variety of ways and also has a good application prospect as an anti-tumor drug [19].

In contrast to necrosis, apoptosis is the most common form of cellular physiological death (non-pathological cell death) [20], which can occur in various stages of embryonic development, tissue reconstruction, immunoregulation, and tumor regression [21]. Caspase-3 is a protease containing 277 amino acid residues, with a molecular weight of about 32 kDa [22], and it is one of the most important terminal shear enzyme of the caspase family in cell apoptosis [23]. Its downstream factor, Bcl-2, is an oncogene that inhibits apoptosis. Currently, the Bcl-2 protein family can be divided into 2 types according to function [24]. One type, similar to Bcl-2, can inhibit apoptosis such as Bcl-XL, Bcl-W, Mcl-1, and A1. While the other type can promote apoptosis, such as BAX, Bcl-Xs, Bik/Nbk, and Bid [25]. Our research showed that RAA-11 can promote the expression of apoptotic initiator caspase-3 and pro-apoptotic factor BAX, and inhibit the expression of apoptosis factor Bcl-2. Therefore, we deem that RAA-11 can induce the apoptosis of human gastric cancer SGC-7901 cells.

In tumor cells, EGFR promotes growth by promoting the proliferation and inhibiting the apoptosis of tumor cells [26]. Furthermore, numerous studies exhibited that EGFR is closely related to inflammation. Mu et al. found that epidermal growth factor receptor 2 (HER2) overexpression was observed at a higher rate in inflammatory breast cancer compared with noninflammatory breast cancer [27]. Li et al. report on in vitro and in vivo studies revealed that berberine inhibits the development of colitis-associated colorectal cancer by interfering with inflammatory response-driven EGFR signaling in tumor cell growth [28]. It has been confirmed that EGFR/PI3K/Akt is an important signal pathway in regulating tumor cell proliferation and apoptosis [29]. Once the EGFR combines with ligand, the PI3K/Akt, Ras/Raf/MEK/ERK signal pathways can be activated through cascade reaction [30]. In the PI3K/Akt pathway, multiple growth factors are involved in signal transduction [31], which is considered as the primary pathway for cancer cell survival by researchers globally [32]. EGFR can activate the PI3K/Akt signal pathway by reducing apoptosis and stimulating protein activity [33]. Chun et al. found that in the MDA-MB-231 cells of breast cancer, EGF can stimulate EGFR autophosphorylation and promote cell growth by activating the PI3K/Akt and MAPK signaling pathways [34].

The Akt gene encodes a 56 kDa serine/threonine protein kinase, and locates in the core of PI3K/Akt signaling pathway [35]. The overactivation of the PI3K/Akt signaling pathway can promote cell proliferation by inhibiting the pro-apoptotic pathway, thereby promoting tumor growth [36]. In addition, Akt can regulate the downstream nuclear transcription factor (NF-κB) through mammalian target of rapamycin (mTOR) [37]. NF-κB further regulates a variety of cancer cell effects, including proliferation, apoptosis, metastasis and angiogenesis [38].

As a transcription factor, NF-κB can control tumorigenesis and resist to cancer treatment by regulating a series of genes related to cell proliferation and apoptosis. It can also regulate the apoptosis and cell proliferation, thereby promoting biological behaviors such as tumor angiogenesis, metastasis and invasion. Furthermore, it participates in physiological and pathological processes such as immunity, inflammation and stress response [39,40]. NF-κB is a crucial mediator of inflammatory and immune responses and a number of phytochemicals that can suppress this immune-regulatory transcription factor are known to have promising anti-inflammatory potential. Liu et al. found that harmine might exert the anti-inflammatory effect by inhibition of the NF-κB signaling pathway [41]. Park et al. thought that inhibiting NF-κB signaling had potential therapeutic applications in cancer and inflammatory diseases [42]. Many studies have suggested that NF-κB is closely related to cancer and plays a key role in the occurrence and development of cancer [43]. Sandner et al. found that in gastric cancer tissue and gastric cancer cells, NF-κB is highly expressed, its activation can not only promote the generation of the chemokine, reactive oxygen species (ROS), prostaglandins, matrix metalloproteinase (MMPs) [44], but it can also alter the phenotype of gastric mucosa cells, involve in cell proliferation, angiogenesis, and cancer cell resistance to apoptosis. Moreover, NF-κB is also related to the prevention, occurrence, development, metastasis, infiltration, treatment and prognosis of gastric cancer [45]. Many studies have proven that the abnormal activation of NF-κB signal pathway is closely related to the occurrence and development of various diseases, such as gastric cancer, colon cancer, ulcerative colitis, and helicobacter pylori related gastritis [46–48]. At the same time, our data showed that RAA-11 significantly downregulated the expression of pathway proteins EGFR, Akt, p-Akt, and NF-κB. Therefore, we deem that RAA-11 inhibits the growth and proliferation of SGC-7901 cells and induces apoptosis through the EGFR/Akt/NF-κB pathway.

Conclusions

RAA-11 can effectively inhibit the growth, proliferation and colony forming ability and induce apoptosis of human gastric cancer SGC-7901 cells, whereby the apoptosis mechanism is related to the EGFR/Akt/NF-κB pathway. However, further in-depth studies and preclinical animal studies are needed to assess the exact mechanism of RAA-11 inducing apoptosis of human gastric cancer SGC-7901 cells, so as to draw scientific theoretical basis for the development and application of RAA-11.