Fifteen immunodeficient female BALB/C nude mice, 5– 6 weeks old were purchased from the Experimental Ani- mal Center of the Henan province, China, They were bred under aseptic conditions and maintained at con- stant humidity and temperature, according to standard guidelines under a protocol approved by Zhengzhou University. Mice in the different groups were subcutane- ously injected in the dorsal scapular region with the BGC-823cells. After transplantation, the skin was closed and the mice were divided randomly into three groups (five mice per group). In 2 days, AST (100 mg/kg body weight) (Group1) and AST (200 mg/kg body weight) (Group2)  were administered once a day. PBS was administered as the control group. Primary tumors were allowed to develop for 28 days. The tumors formed were measured with a caliper every 7 days, and tumor volume was calculated using the formula: volume = π(length × width 2 )/6. Tumors were harvested after 4 weeks.
Abstract: Baicalin is one of the main bioactive flavone glucuronides, which is considered as a medical herb with anticancer activity. However, no detailed studies have ever been reported on its anticancer action through NF-κB/ Snail signaling pathway. This study aimed to investigate the effects of baicalin on the invasion and migration of gas- tric cancer BGC-823cells and epithelial-mesenchymal transition (EMT). Different concentrations of baicalein were used to treat with BGC-823cells. MTT colorimetry was used to observe the inhibitory rate of cell proliferation. The effects of baicalein on the invasion and migration ability of gastric cancer BGC-823cells were determined by cell invasion and migration assay. The roles of baicalein on EMT of BGC-823cells were investigated by examining the protein levels of EMT related markers in BGC-823cells after baicalein and/or TGF-β1 treatments by western blot. Finally, the role of baicalein in TGF-β1-mediated activation of NF-κB/Snail signaling pathway was investigated by ex- amining the protein levels of the factors involved in NF-κB/Snail signaling pathway in BGC-823cells after baicalein and/or TGF-β1 treatment by western blot. Our results have shown that baicalein not only inhibited the proliferation of BGC-823cells, but also reduced the invasion and migration ability of those cells. Baicalein also inhibited EMT of BGC-823cells and TGF-β1-mediated activation of NF-κB/Snail signaling pathway by regulating the expression of the related factors. As a result, baicalein could inhibit EMT and suppress the invasion and migration of human gastric cancer BGC-823cells through NF-κB/Snail signaling pathway.
BGC-823cells were seeded in 10 cm culture dish at the density of 2 × 10 6 cells/dish in 10 mL medium. Cells were treated by costunolide with different concentration (0, 5, 10, 15, 20 and 25 μM) for 24 h. Cells were treated RIPA lysis buffer containing protease and phosphatase inhibitor for 10 min. Total protein was collected by cen- trifuging (12,000×g, 10 min, 4 °C) and mixed with the same volume of sample buffer. Extracted protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane. PVDF membrane was blocked and incubated with rabbit anti- human primary antibody (Bax, Bcl-2, caspase 9, caspase 7, caspase 3, PARP and β-actin, all antibodies were di- luted by 1:1000) overnight at 4 °C. The membrane was washed and incubated with the Dylight 800-labeled goat anti-rabbit fluorescence secondary antibody for 1 h. The membrane was scanned by the Odyssey infrared imaging system (LI-COR Biosciences, Nebraska, USA).
AGS and BGC-823cells were transfected with 50 nm si-circ_0066444 or si-NC. When cell confluence reached approximately 80% at 24 h posttransfection, wounds were created in confluent cells using a 1 mL pipette tip. Cells were then rinsed with media to remove any free-floating cells and debris. Medium was added, and culture plates were incubated at 37 ° C. Wound healing was observed at different time points within the scrape line, and representative scrape lines were photographed. Duplicate wells for each condition were exam- ined, and each experiment was repeated in triplicate.
Dysosma versipellis (Hance) M. Cheng, an important medicinal plant species, is considered ‘endangered’ by the China Species Red List and has been considered as vulnerable by the IUCN due to its rapid decline [42,43]. Therefore, studies on the chemical constituents from D. versipellis and their biological activities have assumed significance for the rational development and utilization of this plant. In our study, fifteen compounds were extracted and identified from D. versipellis grown in Guizhou province, and the cell growth inhibition effects of these constituents on PC3, Bcap-37, and BGC-823cells were carried out by MTT assay. Among these com- pounds, PTO, which was extracted from D. versipellis for the first time, together with DDPT showed potent activities on PC3, Bcap-37, and BGC-823cells in a dose-dependent manner. And the IC 50 values of PTO
BALB/c-nude mice were injected subcutaneously into the right flanks with BGC-823, YAP1-shRNA BGC- 823, MKN45, GES-1, and GES-1-YAP1 cells, and let to grow for 4 weeks to establish the heterotopic xenograft tumor mice model. Results showed that silencing YAP1 significantly inhibited tumor growth. The tumor volume and tumor weight in the YAP1 shRNA BGC-823 group was smaller than those in the BGC-823 group (both P < 0.05, Figure 4A, 4B, 4H and 4I). IHC showed that the YAP1 protein expression in the YAP1 shRNA BGC-823 group was lower than that in the BGC-823 group (Figure 4J and 4K). YAP1 mRNA levels in the YAP1 shRNA BGC-823 group was significantly lower than that in the BGC-823 group (P < 0.05, Figure 4M). All three mice in the MKN-45 group displayed tumors 28 days after cell inoculation (Figure 4C), but IHC showed that MKN-45 cells did not express YAP1 (Figure 4L). Mice injected subcutaneously with GES-1 cells and GES-1 cells overexpressing YAP1 did not generate tumors (Figure 4D and 4E). BGC-823 or YAP1shRNA BGC-823cells were injected into nude mice through the tail vein. Stable knockdown of YAP1 reduced the number of lung metastases (P < 0.05, Figure 4F, 4G, 4O, 4P, and 4N).
Our current results presented the novel NF- κ B inhibitor named Da0324, an asymmetric MAC, which displayed tar- get selectivity for gastric cancer cells. Da0324 significantly inhibited the proliferation of SGC-7901 and BGC-823 gastric cancer cells (Table 1) that had a high level of p65 protein and low expression of I κ B- α protein (Figure 2). Compared with curcumin and BMS345541, Da0324 exhibited better activity against SGC-7901 and BGC-823cells. Da0324 also possessed excellent antitumor activity similar to the active symmetric MACs, EF24, and B19. In addition, Da0324 showed poor inhibition on the normal gastric mucosa epithe- lial cell line GES-1 which had a low level of p65. However, EF24 and B19 significantly inhibited the proliferation of GES-1 cell, which suggested that symmetric MACs were extremely toxic to normal cells. Da0324 significantly inhib- ited the TNF- α -induced NF- κ B activation by suppressing the phosphorylation of IKK and I κ B- α , inhibiting the degrada- tion of I κ B- α , and limiting the NF- κ B subunit p65 nuclear translocation in gastric cancer cell lines. The inhibitory effect of Da0324 corresponded to that of the IKK-specific inhibitor BMS345541 (Figures 3 and 4). All of these results suggested that the antiproliferative effect of Da0324 may be related to the inhibition of NF- κ B signaling pathways, such that Da0324 displayed target selectivity for tumor cells.
miR-135a in BGC-823cells inhibited the expression of phospho-p65 and increased the expression of I κ B α (Figure 4A). Luciferase reporter assay also verified that miR- 135a suppressed the NF- κ B reporter activity (Figure 4B). qPCR detection of the related targets of NF- κ B signaling in the aspect of metastasis controlling confirmed that miR-135a inhibited the mRNA levels of MMP2, MMP9, ICAM-1, and VCAM-1 (Figure 4C). Furthermore, inhibiting endogenous miR-135a in SGC-7901 cells had the opposite effects as revealed by Western blot, luciferase reporter, and qPCR assays mentioned earlier (Figure 4D–F). Hence, we proposed that miR-135a inhibits NF- κ B pathway through directly targeting TRAF5, since TRAF5 could activate this pathway.
Gastric cancer (GC) is among the most malignant cancers with high incidence and poor prognoses worldwide as well as in China. dCTP pyrophosphatase 1 (DCTPP1) is overexpressed in GC with a poor prognosis. Given chemotherapeutic drugs share similar structures with pyrimidine nucleotides, the role of DCTPP1 in affecting the drug sensitivity in GC remains unclear and is worthy of investigation. In the present study, we reported that DCTPP1-knockdown GC cell line BGC-823 exhibited more sensitivity to 5-fluorouracil (5-FU), demonstrated by the retardation of cell proliferation, the increase in cell apoptosis, cell cycle arrest at S phase and more DNA damages. Multidrug resistance 1 (MDR1) expression was unexpectedly down-regulated in DCTPP1-knockdown BGC-823cells together with more intracellular 5-FU accumulation. This was in large achieved by the elevated methylation in promoter region of MDR1 gene. The intracellular 5-methyl-dCTP level increased in DCTPP1-knockdown BGC-823cells as well. More significantly, the strong correlation of DCTPP1 and MDR1 expression was detectable in clinical GC samples. Our results thus imply a novel mechanism of chemoresistance mediated by the overexpression of DCTPP1 in GC. It is achieved partially through decreasing the concentration of intracellular 5-methyl-dCTP, which in turn results in promoter hypomethylation and hyper-expression of drug resistant gene MDR1. Our study suggests DCTPP1 as a potential indicative biomarker for the predication of chemoresistance in GC.
In addition, cell cycle arrest also plays an important role in the upstream fac- tors that induce apoptosis. A number of reports have revealed that PN can cause apoptosis by inducing cell cycle arrest . The PI single-staining experiment showed that the proportion of G1 phase of BGC-823cells in PN treatment group was significantly increased, and the proportion of S phase was significantly de- creased. This indicates that PN can block the cell cycle of BGC-823 in G1 phase. We further examined the expression of cell cycle-associated proteins. When the chromosomal DNA of the cell is damaged, P53 binds to the corresponding part of the DNA and acts as a special transcription factor , which activates p21 transcription . In the present study, we found that the total protein levels of BGC-823 cell cycle inhibitory regulatory proteins P21 and P53 are elevated after PN treatment, and multiple Cyclin-CDK complexes bind to P21 and inhibit the corresponding protein kinase activity . At the same time, the experimental results showed that the total protein levels of the cell cycle-promoting regulatory proteins CyclinD1 and CyclinE1 were decreased, which also proved that the treatment of PN caused G1 arrest of its cell cycle.
Cell proliferation plays important role in cancer develop- ment. We then investigated the anti-proliferative effect of gramicidin on human gastric cancer cells and colony formation assay was used. As shown in the Fig. 2a, cells were treated with gramicidin at various concentration for 10 days and the colony formation rate of SGC-7901 and BGC-823cells decreased significantly. Quantitative analy- sis of the clone formation rate showed that gramicidin sup- pressed proliferative capacity of SGC-7901 and BGC-823cells in a concentration-dependent manner (Fig. 2b, c). However, the proliferation of human gastric mucosal epi- thelial cells GES-1 was not affected by gramicidin when compared to the control group (Fig. 2d). Only when the concentration of gramicidin reached to 40 nM, the prolifer- ation of the GES-1 cells was inhibited (P < 0.05). The above results suggested that the gramicidin could inhibit the pro- liferation of the gastric cancer cells SGC-7901 and BGC- 823. As SGC-7901 showed a more sensitive pattern upon gramicidin treatment, we next evaluate further anti-tumor effect of gramicidin on GC using the SGC-7901 cells.
To further investigate whether circNRIP1 regulates hypoxia-induced 5-FU resistance in GC cells through HIF-1 α -dependent glycolysis, BGC-823cells were trans- fected with Lv-NRIP1 or Lv-ctrl, followed by treatment with 2-DG under a hypoxic condition. As displayed in Figure 7A and B, transfection of Lv-NRIP1 markedly elevated the mRNA level of MDR1 and the protein level of p-gp in BGC-823cells exposed to hypoxia, while cotreatment with Lv-NRIP1 and 2-DG strikingly recuper- ated MDR1 mRNA level and p-gp protein level. In addi- tion, glucose consumption and lactate production were conspicuously increased following Lv-NRIP1 transfection in BGC-823cells exposed to hypoxia, which was mark- edly conversed by 2-DG treatment (Figure 7C and D). Simultaneously, the levels of G6P were substantially increased in BGC-823cells transfected with Lv-NRIP1 under a hypoxic condition, however, circNRIP1-mediated increase of G6P levels was markedly abolished by 2-DG treatment (Figure 7E). Furthermore, a marked elevation of HIF-1 α was discovered in BGC-823cells transfected with Lv-NRIP1 under a hypoxic condition, and this elevation was reversed by 2-DG treatment (Figure 7F). Besides, upregulation of circNRIP1 reduced the sensitivity of BGC-823cells to 5-FU accompanied by the increased cell survival, and this action was marked overturned by 2-DG administration (Figure 7G).
17 miRNAs were predicted as the potential targets of circCACTIN by the circular RNA Interact- ome Database. Firstly, 5 miRNAs with scores above 90 including hsa-miR-1205, hsa-miR-1225-3p, hsa-miR- 1233, hsa-miR-331-3p, hsa-miR-661 were selected. QRT-PCR was used to detect the expression changes of these miRNAs in the circCACTIN down-regulation BGC-823cells. The results indicated that hsa-miR-331-3p expression was most significantly affected by circCACTIN (Fig. 4A). Dual-luciferase reporter assays were performed to verify if there was a binding site between circCACTIN and miR-331-3p. The alignments of potential binding site and mutant type of circCACTIN were constructed through bioinformatics analysis (Fig. 4B). The firefly luciferase reporter activity was significantly decreased in the group of co-transfection of hsa-miR-331-3p mimics and circCACTIN WT in BGC-823cells, and the circCACTIN mutant group showed no notable change of luciferase reporter activity (Fig. 4C). This finding confirmed that binding interaction existed between circCACTIN and miR-331-3p.
The assessment of the ability of exosomal miR-221 to regulate adhesion of GC cells to the matrix was conducted using adhesion assays. Consequently, threefold of BGC-823cells cultured with exosomes that were treated with miR-221 mimics successfully adhered to the matrix compared with the number in the NC group (Figure 4F). Similar results were observed in SGC-7901 cells. In contrast, the number of adherent GC cells was significantly decreased after culture with exosomes transfected with miR-221 inhibitor. In conclusion, exosomes with high expression of miR-221 can effectively promote GC cell migration, invasion, and adhesion to the matrix. Therefore, a sufficient amount of miR-221 mimics and inhibitor existed in exosomes and effectively contributed to the oncogenic activity of GC cells. These upregulated biological properties of GC cells triggered by miR-221 mimics transfected into exosomes may be a new mechanism that explains the frequent metas- tasis of GC. According to these results, it is not difficult to understand why the high expression of exosomal miR-221 in peripheral blood is positively correlated with the poor clinical prognosis of GC.
effects of MIAT on the cell cycle and apoptosis of gastric cells by flow cytometry. Compared with si- control, si-MIAT-1 and si-MIAT-2 led to an increased proportion of BGC-823cells in the S phase, but a decreased proportion of cells in G0/G1 phase and G2/M phase (Fig. 2c), indicating that S-phase arrest may be a mechanism of MIAT depletion-induced growth inhibition. Similar results were observed in MGC- 803 cells (Additional file 2: Figure S2A). Furthermore, the rate of apoptotic cells in si-control, si-MIAT-1 and si- MIAT-2 transfected BGC-823cells were 5.47%, 27.7% and 25.33%, respectively (Fig. 2d). Similar results were observed in MGC-803 cells (Additional file 2: Figure S2B). These re- sults suggested that MIAT depletion may inhibit the growth of GC cells by inducing S-phase arrest and apoptosis.
GRINA regulates apoptosis via Bcl-2 family members Studies have shown that GRINA could participate in biological processes such as apoptosis and had a close relationship with Bcl-2 family members . Therefore, we examined apoptosis in the GRINA-shRNA and con- trol groups by the Annexin V/PI double labelling method. The percentages of apoptotic cells were obvi- ously augmented in shRNA groups compared with those in control groups after cells were cultured in medium without foetal bovine serum (FBS) for 48 h (Figs 7A-7B). TUNEL staining also reaveled that mice tumor tissues in the shRNA group showed higher apoptosis rate than those in the control group (Fig. 7C). Then we assessed Bcl-2 and Bax expression after GIRNA knockdown in AGS and BGC-823cells. We found that Bcl-2 expres- sion was significantly decreased whereas Bax expression was obviously increased in both cell lines (Fig. 7D). The levels of Cleaved-caspase3, Cleaved-caspase7, and Cleaved-caspase9 were significantly increased (Fig. 7D). These results suggested GRINA could regulate apoptosis via Bcl-2 family members.
Abstract: Introduction: Previous studies have shown that the dysregulation of miRNAs are frequently associated with cancer progression. Deregulation of miR-211 has been observed in various types of human cancers. However, its biological function in gastric cancer (GC) is still unknown. Methods: The expression of miR-211 in GC was detected by using quantitative real-time PCR (qRT-PCR). The miR-211 mimics and inhibitor were designed and transfected into BGC-823cells. Then, we explore the probable biological function of miR-211 in gastric cancer cell proliferation and invasion in vitro. A luciferase reporter assay and western blot were performed to confirm the target gene of miR- 211. Results: MiR-211 was significantly down-regulated in GC. Over-expression of miR-211 inhibited gastric cancer cell proliferation and invasion in vitro, conversely, down-regulated expression of miR-211 promoted gastric cancer cell proliferation and invasion. In addition, the sex-determining region Y-related high mobility group box 4 (SOX4) is identified as a target of miR-211 in GC cells, and SOX4 expression levels was inversely correlated with miR-211. Furthermore, knockdown of Sox4 inhibited the proliferation and invasion in GC cells. Conclusion: miR-211 could in- hibit GC cell proliferation and invasion partially by down-regulating SOX4. MiR-211 might be a potential therapeutic target for GC treatment in the future.
The homotypic-mediated internalization path- way of HepM-PLGA by HepG2 cells was further investigated (Figure 3). The fluorescence intensity enhances with time, indicating the time-dependent internalization pathway of HepM-PLGA by HepG2 cells. To further verify the homotypic aggregation of cancer cells and the homologous targeting ability of HepM-PLGA, certain kinds of cancer cells, including human gastric carcinoma cells (BGC-823cells), cervical carcinoma cells (HeLa cells) and breast cancer cells (MCF-7 cells), were incubated with FITC-HepM-PLGA. The CLSM images and the flow cytometric assay exhibit FITC-HepM-PLGA with a unique homologous targeting ability to HepG2 cells rather than other kinds of cancer cells (Figure 4 & S4). Similarly, the FITC-MCFM-PLGA nanoparticles could only recognize MCF-7 cells rather than other cancer cells (Figure S5 & S6). Besides, in consideration of other cell membranes including red blood cell (RBC), white blood cell (WBC) and platelet (PLT) were used for nanoparticle functionalization as well [44-46], the unique capabilities of FITC-HepM-PLGA was compared with PLGA nanoparticles coated by RBC membranes (FITC-RBCM-PLGA). The FITC-RBCM- PLGA nanoparticles were incapable of targeting tumor cells specifically (Figure S7). Taken together, the HepM-PLGA nanoparticles exclusively target
Figure 7: Blockage of STAT3 activation reverses GRIM-19 loss-driven tumor growth in vitro and in vivo. A,B. Inhibition of STAT3 reduced GRIM-19 loss-induced STAT3 activation and STAT3-responsive genes expression. SGC-7901-shGR19 cells were treated with a pool of STAT3 siRNAs (siSTAT3) or STAT3 inhibitor S3I-201. Phospho-STAT3(Y705) and total STAT3 protein were detected by Western blot (A) and STAT3-responsive targets were analyzed with quantitative RT-PCR (B). GAPDH was used as an internal control. C, D, E. Abrogation of STAT3 activation reversed GRIM-19 loss-driven cell proliferation and colony formation in vitro. SGC-7901-shGR19 cells were treated with siSTAT3 or S3I-201. Cell viability was detected by WST-1 assay at absorbance 450nm for 3 consecutive days (C) and colony formation ability was determined by colony formation assay (D, E). Data are presented as mean ± SD of 3 independent experiments. Representative images of colonies formed are shown. F, G. Inhibition of STAT3 activation reversed GRIM-19 loss-derived tumorigenesis in vivo. Xenograft mouse models from SGC-7901-shGR19 were established as described in Materials & Methods. When tumor volumes had reached approximately 50 mm 3 , Mice (n=8 mice/group) were intratumorally injected four times with or without S3I-201, and tumor volume was calculated every two
on tissues from 35 patients showed that ECRG4 mRNA was downregulated in GC tissues ( p,0.05; Figure 1B). Using an IHC assay, we also observed a lower expression inten- sity of the ECRG4 protein in GC tissues from 102 patients (Figure 1C). Among the 102 GC patients, 35.3% (38/102) showed a lower expression of ECRG4 in GC tissues, while 3.9% (4/102) showed a lower expression of ECRG4 in normal tissues (p,0.01). The ECRG4 protein was also related to the differentiation state of GC. As shown in Figure 1C, lower ECRG4 expression was always associated with poorer dif- ferentiation. To further confirm the ECRG4 expression in cells, we performed WB and qRT-PCR using four GC cell lines. Both the assays demonstrated that protein and mRNA expression of ECRG4 was downregulated in all GC cell lines compared to that in a normal cell line (Figure 1D and E).