HGC-27 Cells Research Articles

Celastrol Induces Necroptosis and Ameliorates Inflammation via Targeting Biglycan in Human Gastric Carcinoma.

Celastrol, a triterpene isolated from the root of traditional Chinese medicine Thunder of God Vine, possesses anti-cancer and anti-inflammatory activity to treat rheumatoid disease or as health product. Necroptosis is considered as a new approach to overcome chemotherapeutics resistance. However, whether celastrol exerts necroptosis leading to gastric cancer cell death is still unclear. Here, for the first time we showed that celastrol induced necroptosis in HGC27 and AGS gastric cancer cell lines. More importantly, celastrol down-regulated biglycan (BGN) protein, which is critical for gastric cancer migration and invasion. Furthermore, celastrol activated receptor-interacting protein 1 and 3 (RIP1 and RIP3) and subsequently promoted the translation of mixed-lineage kinase domain-like (MLKL) from cytoplasm to plasma membrane, leading to necroptosis of gastric cancer cell, which was blocked by over-expression BGN. In addition, celastrol suppressed the release of pro-inflammatory cytokines TNF-α and IL-8 in HGC27 and AGS cells, which was reversed by over-expression BGN. Taken together, we identified celastrol as a necroptosis inducer, activated RIP1/RIP3/MLKL pathway and suppressed the level of pro-inflammatory cytokines by down-regulating BGN in HGC-27 and AGS cells, which supported the feasibility of celastrol in gastric cancer therapy.

Oridonin induces apoptosis in HGC-27 cells by activating the JNK signaling pathway.

Gastric cancer (GC) is a very common type of cancer. Although current treatment modalities include surgical resection and chemotherapy, many patients are either not eligible for radical resection or have a poor response to chemotherapy. Due to the complex features of the disease, there is a need for complementary therapy. In the present study, the effects of oridonin on cell proliferation, invasion and apoptosis were assessed in the HGC-27 cell line using the Cell Counting Kit-8 assay, real-time cell analysis, and an Annexin V-FITC/propidium iodide (PI) detection kit, respectively. The effect of oridonin on apoptosis, through the JNK pathway, was also investigated using western blotting. The present study demonstrated that oridonin can suppress cell viability and inhibit cell proliferation by inducing G2/M arrest. Oridonin also induced caspase-dependent apoptosis in cells by activating the phosphorylated-JNK/C-JUN pathway. These results demonstrate the potential of oridonin as a potential therapeutic compound for the treatment of GC.

FAM60A, increased by Helicobacter pylori, promotes proliferation and suppresses apoptosis of gastric cancer cells by targeting the PI3K/AKT pathway

Helicobacter pylori (H. pylori) infection can promote the development of gastric cancer (GC); however, the underlying mechanism is not clear. FAM60A has been found showing high levels in some cancer cells, including lung cancer (A549), and pancreatic cancer (Capan-2) cell lines. Data in oncomine showed that FAM60A overexpression was an critical prognostic factor in GC. In this study, we showed that knockdown of FAM60A could revert the increase of proliferation and the decrease of apoptosis caused by H.pylori infection in HGC-27 and AGS cells. Conversely, FAM60A upregulation promoted proliferation and inhibited apoptosis in HGC-27 and AGS cells. We also found that the PI3K/AKT pathway inhibitor LY294002 could revert the changes caused by FAM60A upregulation in HGC-27 and AGS cells. Thus, our study provides evidence that FAM60A act as a carcinogen and suggests that H. pylori-induced upregulation of FAM60A may contribute to the development of gastric cancer.

Circ-DB participates in the progression of gastric cancer by competitively binding microRNA-34a to regulate the expression of MET.

To figure out the possible role and mechanism of circ-DB in the pathogenesis of gastric cancer (GCa). 32 cases of postoperative GCa tissue samples and adjacent ones were collected and divided into groups of ≥ 5 cm and < 5 cm according to tumor diameter. Circ-DB, microRNA-34a, and MET expressions were detected by real time-quantitative PCR (RT-qPCR) in GCa tissues and adjacent tissues. To determine the main mode of action of circ-DB, the subcellular localization of circ-DB was examined by dividing the cells into the nucleus and cytoplasmic fractions. The binding of microRNA-34a to circ-DB was demonstrated by a Dual-Luciferase reporter gene assay. The expression of circ-DB in HGC-27 and AGS cells was overexpressed and knocked down to evaluate the migration function of the cells by transwell. The protein expression of MET, as well as the target gene of microRNA-34a, was detected by Western blot. The expression of circ-DB and MET in GCa tissues was significantly higher than that in the corresponding adjacent tissues. Circ-DB was positively correlated with MET expression, while microRNA-34a expression was negatively correlated with circ-DB and MET expression. Circ-DB was mainly located in the cytoplasm, and the Dual-Luciferase reporter gene demonstrated that microRNA-34a can bind to circ-DB. The down-regulation of circ-DB expression inhibited the migration of HGC-27 and AGS cells. In vitro cell experiments showed that low expression of circ-DB inhibited cell migration, which could be recovered by the co-transfection with microRNA-34a inhibitor. Circ-DB may regulate MET level through microRNA-34a and affect the proliferative ability and migration of GCa cells.

Hsa_circ_0001546 acts as a miRNA-421 sponge to inhibit the chemoresistance of gastric cancer cells via ATM/Chk2/p53-dependent pathway

Circular RNAs (circRNAs) are a new class of noncoding RNAs, play a crucial role in tumor initiation and development. Hsa_circ_0001546 is a novel circular RNA that was downregulated in gastric cancer (GC) tissues, however its function and mechanism in GC has not been studied. Our study verified that circ_0001546 was decreased in GC and correlated with the poor prognosis. Next, Pull-down assay and dual-luciferase reporter assay verified that miR-421 was a target of circ_0001546 while ATM (Ataxia telangiectasia mutated) was target by miR-421. Overexpression of circ_0001546 inhibited the proliferation and chemoresistance of HGC-27 cells, and increased the expression of ATM. In addition, circ_0001546 overexpression reversed the effect of miR-421 overexpression. What is more, circ_0001546 inhibits the chemoresistance of HGC-27 cells to L-OPH (Oxaliplatin) may through the activation of the ATM/checkpoint kinase 2 (Chk2)/p53-dependent signaling pathway. In summary, our study proved that circ_0001546 sponges miR-421 to upregulate the expression level of ATM and inhibit the proliferation and chemoresistance through the activation of the ATM/Chk2/p53-dependent pathway.

Eukaryotic translation initiation factor 3B downregulation inhibits cell proliferation and promotes cell apoptosis through negatively regulating tumor necrosis factor receptor superfamily member 21 in gastric cancer

Background: This study aimed to detect eukaryotic translation initiation factor 3B (EIF3B) expression in gastric cancer (GC) cell lines, and further explore the effect of EIF3B downregulation on GC cell proliferation and apoptosis. Methods: EIF3B mRNA expression and protein expression in human GC cell lines (NCI-N87, AGS, HGC-27, BGC-823 and MGC80-3) and human gastric mucosal epithelial cell line (GES-1) were detected. Control siRNA (Si-NC group) and EIF3B siRNA (Si-EIF3B group) were transfected into NCI-N87 cells. Rescue experiment was performed by transfection of EIF3B siRNA (Si-EIF3B group) and EIF3B siRNA plus tumor necrosis factor receptor superfamily member 21 (TNFRSF21) siRNA (Si-EIF3B & Si-TNFRSF21 group) into NCI-N87 cells. Besides, cell proliferation, apoptosis, TNFRSF21 expression and TRAF1 expression were assessed. Results: EIF3B mRNA expression and protein expression were elevated in NCI-N87, AGS, HGC-27 and BGC-823 cell lines compared to GES-1 cell line. In NCI-N87 cells, proliferation was reduced in Si-EIF3B group compared to Si-NC group. For cell apoptosis, its rate and apoptotic marker C-Caspase 3 expression were increased but anti-apoptosis marker Bcl-2 expression was reduced in Si-EIF3B group compared to Si-NC group. Moreover, mRNA expression and protein expression of TNFRSF21 were increased in Si-EIF3B group compared to Si-NC group, while mRNA expression and protein expression of TRAF1 were reduced in Si-EIF3B group compared to Si-NC group. In rescue experiment, cell proliferation was increased but apoptosis was decreased in Si-EIF3B & Si-TNFRSF21 group compared to Si-EIF3B group. Conclusions: EIF3B is overexpressed in GC cell lines, and its downregulation inhibits cell proliferation while promotes apoptosis through negatively regulating TNFRSF21 in GC.

Differentially expressed long noncoding RNAs and regulatory mechanism of LINC02407 in human gastric adenocarcinoma.

BACKGROUNDLong noncoding RNAs (lncRNAs) have been identified to play important roles in the development and progression of various tumors, including gastric cancer (GC). However, the molecular role of lncRNAs in GC progression remains unclear.AIMTo investigate the differential expression of lncRNAs in human GC and elucidate the function and regulatory mechanism of LINC02407.METHODSThe Cancer Genome Atlas database was used to investigate the involvement of lncRNAs in GC. Quantitative real-time polymerase chain reaction was used to estimate the relative expression level of LINC02407 in GC tissues and cells. Functional experiments including CCK8 assay, apoptosis assay, wound healing assay, and transwell assay were used to investigate the effect of LINC02407 on GC cells. Some microRNAs were predicted and verified via bioinformatics analysis and the luciferase reporter system. Predictive analysis and Western blot assay were used to analyze the expression of related proteins.RESULTSMany differentially expressed lncRNAs were identified in GC, and some of them including LINC02407 can affect the survival. LINC02407 was upregulated in tumor tissues compared with adjacent tissues. HGC-27 cells showed the highest LINC02407 expression and HaCaT cells exhibited the lowest expression. Different experiment groups were constructed using LINC02407 overexpressing plasmids and related siRNAs. The results of functional experiments showed that LINC02407 can promote the proliferation, migration, and invasion of GC cells but inhibit apoptosis. Luciferase reporter assay showed that hsa-miR-6845-5p and hsa-miR-4455 was downstream regulated by LINC02407. Western blot analysis showed that adhesion G protein-coupled receptor D1 (ADGRD1) was regulated by the LINC02407-miR-6845-5p/miR-4455-ADGRD1 pathways.CONCLUSIONLINC02407 plays a role in GC through the LINC02407-miR-6845-5p/miR-4455-ADGRD1 pathways, and thus, it may be an important oncogene and has potential value in GC diagnosis and treatment.

Abstract #4363 Stress perception and mediators following the diagnosis of early-stage malignant melanoma, a comparison group and follow-up study in patients diplaying psychooncologic need

Neurotrophin receptor-interacting melanoma antigen-encoding protein (NRAGE) is a type II melanoma-associated antigen that plays an essential role in various processes, including cell differentiation and apoptosis. NRAGE has been shown to act as a cancer-related protein, with complex and apparently contradictory functions in a variety of cancers. In the current study, we examined the expression of NRAGE protein in 169 gastric cancer samples. NRAGE upregulation was correlated with advanced TNM stage, local invasion, and poor survival. Importantly, NRAGE could serve as an independent prognostic factor in patients with gastric cancer. We also examined the expression of NRAGE protein in GES-1 normal gastric epithelial cells and in six gastric cancer cell lines. Inhibition of NRAGE expression by transfection with small interfering RNA reduced the proliferation and invasion of MGC-803 and HGC-27 cells, as demonstrated by CCK-8 and Matrigel invasion assays. NRAGE depletion also sensitized HGC-27 and MGC-803 cells to cisplatin, as shown by CCK-8 and Annexin V/propidium iodide analyses. Western blot analysis also showed that NRAGE depletion negatively regulated Bcl-2 and p-ERK and upregulated ZO-1 and p27 expression levels. In conclusion, our results suggest that NRAGE acts as a tumor promoter in gastric cancer by facilitating cancer invasion and chemoresistance, possibly through regulation of p-ERK and Bcl-2.

MiR-181d inhibits cell proliferation and metastasis through PI3K/AKT pathway in gastric cancer.

Gastric cancer is the second highest mortality tumor and the fourth most common cancer worldwide that has high aggressiveness. MicroRNA-181d (miR-181d) has been established to be a tumor suppressor, by suppressing cell proliferation, cell cycle, and promoting apoptosis in several cancers. The purpose of this study is to explore the great roles of miR-181d in gastric cancer. The Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot were applied to calculate the mRNA and protein levels of miR-181d and genes. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assays were utilized to measure the proliferative and invasive abilities. The Kaplan-Meier method was conducted to calculate the overall survival of gastric cancer patients. MiR-181d was detected to be downregulated in gastric cancer tissues and cell lines compared to the peritumoral normal tissues and normal cell line. Downregulation of miR-181d predicted poor prognosis of gastric cancer patients. Cylindromatosis gene (CYLD) was overexpressed in gastric cancer tissues, which was confirmed to be a target gene of miR-181d in gastric cancer cell line HGC-27. Moreover, miR-181d inhibited the proliferation through CYLD/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway and inhibited the invasion-mediated epithelial-mesenchymal transition (EMT) in HGC-27 cells. In addition, overexpression of miR-181d suppressed tumor growth and xenograft tumorigenesis of HGC-27 cells in vivo. MiR-181d functioned as a tumor suppressor by inhibiting the proliferation via PI3K/AKT pathway in vitro and in vivo and inhibiting invasion-mediated epithelial-mesenchymal transition (EMT) by targeting CYLD in gastric cancer. The newly identified miR-181d/CYLD axis provides novel insight into the pathogenesis of gastric cancer.

Novel tertiary sulfonamide derivatives containing benzimidazole moiety as potent anti-gastric cancer agents: Design, synthesis and SAR studies

With the expectation to find out new anti-gastric cancer agents with high efficacy and selectivity, a series of novel tertiary sulfonamide derivatives were synthesized and the anti-cancer activity was studied in three selected cancer cell lines (MGC-803, PC-3, MCF-7) in vitro. Some of the synthesized compounds could significantly inhibit the proliferation of these tested cancer cells and were more potent than the positive control (5-Fu). The structure-activity relationship of tertiary sulfonamide derivatives was explored in this report. Among the tested compounds, compound 13g containing benzimidazole moiety showed the best anti-proliferation activities against MGC-803 cells (IC50 = 1.02 μM), HGC-27 cells (IC50 = 1.61 μM), SGC-7901 (IC50 = 2.30 μM) cells as well as the good selectivity between the cancer and normal cells. Cellular mechanism studies elucidated compound 13g inhibited the colony formation of gastric cancer cell lines. Meanwhile, compound 13g arrested cell cycle at G2/M phase and induced cell apoptosis. Mechanistically, compound 13g markedly decreased p-Akt and p-c-Raf expression, which revealed that compound 13g targeted gastric cancer cell lines via interfering with AKT/mTOR and RAS/Raf/MEK/ERK pathways. All the findings suggest that compound 13g might be a valuable lead compound for the anti-gastric cancer agents.

LOW CONCENTRATION OF BISPHENOL A INDUCES PROLIFERATION OF GASTRIC CANCER CELLS, HGC-27

Bisphenol A, an endocrine disrupting compounds that affect human homeostasis. Studies on BPA are focusing on the impact of BPA in reproductive function and brain development. However, the effect of BPA on gut especially gastric cells is not well explored. Gut is directly in contact with ingested BPA; therefore, we aimed to determine the effect of BPA exposure on gastric cells proliferation at safe recommended concentration. Human gastric cancer cells (HGC-27) were treated with BPA at different concentration (low: 10-9M, 10-7M; high10-5M, 10-4M) and time point (24 hr, 48 hr, 72 hr). Cell viability assays were determined using MTS assay. Cells were further stained with Alexa Fluor-635 (F-actin) and Fluorescein (Hif-1α) protein for immunocytofluorescence. Data were analysed using ANOVA (p&lt;0.05, n≥3). Cells treated with 10-9M BPA showed significance increase of cell viability after 48 hr (Mean ±SEM; 146%±0.03, p=0.01) and 72 hr (113%±0.03, p=0.00) compared to 24 hr treatment (77%±0.11, p=0.002). Similarly, cell treated with 10-7M BPA showed a significance increase after 48 hr (141%±0.03, p=0.03) and 72 hr (190%±0.03, p=0.02) compared to 24 hr cells treated with 10-7M (88%±0.05, p=0.01) and untreated (100%±0.07). Lower concentration of BPA increases the condensation of F-actin in all HGC-27 cells. Meanwhile, translocation of Hif-1α protein were observed in all BPA-exposed cells. Findings of this study revealed that BPA induced proliferation and condensation of F-actin structure of gastric cancer cells at low concentration.

Expression of microtubule actin cross-linking factor 1 in gastric carcinoma and its effect on cell invasion and metastasis

Objective To explore the role of microtubule actin cross-linking factor 1(MACF1) in the metastasis of gastric cancer. Methods From 2009 to 2012, at The First Affiliated Hospital of Zhengzhou University, the paraffin blocks of gastric cancer and normal tissue adjacent to cancer of 107 patients who received radical gastrectomy were collected. The expression of MACF1 in tissues at protein level was detected by immunohistochemical staining. In 2017, at The First Affiliated Hospital of Zhengzhou University, fresh specimens samples of gastric cancer and normal tissue adjacent to cancer of 42 patients who received radical gastrectomy were also collected. The expression of MACF1 at mRNA level was determined by quantitative real-time polymerase chain reaction (PCR). MACF1 knockout gastric cell line was established. The effects of MACF1 on cell migration and invasion were verified by wound-healing test and Transwell assay. The effects of MACF1 on cell microtubule and actin were analyzed by filamentous actin (F-actin) staining. T-test, chi-square test and multivariate analysis were used for statistical analysis. Results The positive expression rate of MACF1 in gastric carcinoma tissues was 71.0%(76/107), which was significantly higher than that of the corresponding normal tissues adjacent to cancer (22.4%, 24/107), and the difference was significant (t=4.145, P=0.016). The expression of MACF1 at mRNA level in cancer tissues of 42 patients with gastric cancer was 6.463±0.672, which was significantly higher than that of corresponding normal tissue adjacent to cancer (1.727±0.331), and the difference was statistically significant (t=6.326, P<0.01). The differences in positive expression rate of MACF1 in different tumor infiltration depth, different TNM stage and lymph nodes metastasis were statistically significant (χ2=1.170, 7.959 and 5.288; all P<0.01). The five-year survival rate of patients with high expression of MACF1 was 32.9% (25/76), which was significantly lower than that of patients with normal MACF1 expression (64.5%, 20/31), and the difference was statistically significant (χ2=25.093, P=0.034). The high expression of MACF1 was an independent prognostic factor affecting overall survival rate in patients with gastric cancer after surgery(hazard ratio (HR)=0.513, 95%confidence interval (CI): 0.411 to 0.922, P=0.038). The results of wound-healing assay showed that at 24 hour after wound the migration ability of MACF1 knockout AGS- MACF1-/- cells was (18.77±3.82)%, which was lower than that of wild type AGS cells ((76.24±5.36)%), and the difference was statistically significant (t=6.249, P=0.014). The migration ability of MACF1 knockout HGC27-MACF1-/-cells was (42.48±7.37)%, which was lower than that of wild type HGC27 cells ((82.35±4.28)%), and the difference was statistically significant (t=5.938, P=0.017). The results of Transwell assay indicated that the number of migration cells of MACF1 knockout AGS-MACF1-/- cells was 87.0±11.0, which was less than that of wild type AGS cells (200.0±16.0), and the difference was statistically significant (t=5.820, P=0.028). The number of migration cells of MACF1 knockout HGC27-MACF1-/-cells was 151.0±13.0, which was less than that of wild type HGC27 cells (268.5±20.5), and the difference was statistically significant (t=4.840, P=0.040). The results of F-actin staining demonstrated that the number of actin filaments of MACF1 knockout AGS-MACF1-/- cells was 216.60±18.09, which was less than that of wild type AGS cells (491.30±5.02), and the difference was statistically significant (t=14.630, P=0.005). Conclusions The abnormally high expression of MACF1 in gastric cancer tissues may be correlated with the poor prognosis of patients with gastric cancer. MACF1 promotes the invasion and metastasis of gastric cancer cells by affecting the formation of F-actin and cell skeleton. Key words: Stomach neoplasms; Actins; Invasion and metastasis; MACF1; Cell skeleton; CRISPR/Cas9

MiR-99a suppresses cell migration and invasion by regulating IGF1R in gastric cancer.

Gastric cancer is a common kind of gastrointestinal malignancies. Increasing evidence indicates dysregulation of microRNA-99a (miR-99a) in gastric cancer, and has been extensively investigated in terms of cancer formation, progression, diagnosis, therapy, and prognosis. The purpose of this study is to explore how miR-99a worked in gastric cancer on migration and invasion. The mRNA and protein levels of miR-99a and insulin-like growth factor 1 receptor (IGF1R) in gastric cancer were measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. Transwell assay was employed to analyze the migratory and invasive capacities. The Dual-Luciferase reporter assay was performed to confirm miR-99a mediated the expression of IGF1R by directly targeting its mRNA 3'-untranslated regions (3'-UTR) in gastric cancer cells. MiR-99a was discovered to be significantly downregulated while IGF1R was upregulated in gastric cancer tissues and cell lines. The expression of miR-99a had a negative correlation with the IGF1R expression in gastric cancer tissues. Moreover, miR-99a was low expressed in gastric cancer cells HGC-27 and MGC-803 compared to the normal cell line. MiR-99a suppressed the migration and invasion through directly binding to the 3'-UTR of IGF1R mRNA in HGC-27 cells. In addition, IGF1R could reverse partial roles of miR-99a on migration and invasion in gastric cancer. MiR-99a inhibited the migratory and invasive abilities by regulating the expression of IGF1R. MiR-99a was downregulated while IGF1R was upregulated in gastric cancer cell lines. The newly identified miR-99a/IGF1R axis provides novel insight into the pathogenesis of gastric cancer.

Type II cGMP-dependent protein kinase phosphorylates EGFR at threonine 669 and thereby inhibits its activation

Our previous study demonstrated that type II cGMP-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF) induced tyrosine phosphorylation/activation of the EGF receptor (EGFR). This paper was designed to investigate the mechanism of the inhibition of PKG II on EGFR activation. Gastric cancer cells HGC-27 and AGS were infected with an adenoviral vector encoding the cDNA of PKG II (Ad-PKG II) to overexpress PKG II and treated with 8-(4-chlorophenylthio) guanosine-3′,5′-cyclic monophosphate (8-pCPT-cGMP) to activate the kinase. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assay were performed to detect the interaction between PKG II and EGFR. Western blotting, mass spectrometry (MS) and site mutagenesis were performed to detect the PKG II-specific phosphorylation site on EGFR. The results showed that in living COS-7 cells, which were infected with Ad-PKG II and treated with 8-pCPT-cGMP, there was an interaction between PKG II and EGFR. The results also showed that PKG II caused threonine 669 (T669) phosphorylation of EGFR in HGC-27 and AGS cells infected with Ad-PKG II and treated with 8-pCPT-cGMP, and then inhibited the activation of EGFR. When T669 of EGFR was mutated to alanine, the inhibitory effect of PKG II on the activation of EGFR was eradicated. These findings suggested a PKG II-specific phosphorylation site on EGFR, and might be beneficial to illuminate the anti-tumor role of PKG II.

Cytotoxicity of lanostane-type triterpenoids and ergosteroids isolated from Omphalia lapidescens on MDA-MB-231 and HGC-27 cells

Omphalia lapidescens Schroet. is a medicinal macrofungus in China that has shown good antitumor activity in recent research. To explore the potential cytotoxic compounds from O. lapidescens, the ethyl acetate soluble fraction of a 95% ethanol extract with cytotoxic activity was phytochemically investigated. A new tetranorlanostane triterpenoid (1) and a new ergosteroid (4), together with two known lanostanes (2 and 3) and six known Δ22-ergosteroids (5−10), were isolated. Notably, compound 1 possesses a new 24,25,26,27-tetranorlanostane carbon skeleton. All isolates, except compound 8, were tested for cytotoxic activities using a human breast cancer cell line (MDA-MB-231) and a human gastric cancer cell line (HGC-27) in vitro. The new nortriterpenoid (1) exhibited no obvious cytotoxicity, while the known triterpenoid (3) showed significant cytotoxicity against MDA-MB-231 and HGC-27 cells. Compound 4 exhibited the highest cytotoxicity against MDA-MB-231 and HGC-27 cells, with IC50 values of 11.33 ± 2.18 and 12.28 ± 3.64 μM, respectively. Furthermore, Hoechst fluorescence 33342 staining and Western blot tests demonstrated that compound 4 induced apoptosis in MDA-MB-231 cells by downregulating procaspase-3 expression and upregulating the expression ratio of Bax/Bcl-2.

Polyphyllin I induces autophagy and cell cycle arrest via inhibiting PDK1/Akt/mTOR signal and downregulating cyclin B1 in human gastric carcinoma HGC-27 cells

Paris polyphylla. is a traditional medicinal herb that has long been used to prevent cancer in many Asian countries. Polyphyllin I (PPI), an important bioactive constituent of Paris polyphylla, has been found to exhibit a wide variety of anticancer activities in many types of cancer cells. However, the effects of PPI on human gastric carcinoma cells and its mechanism of action remain unclear. In this study, we examined the effective anti-gastric carcinoma activity of PPI and its underlying mechanism of action in HGC-27 cells. In vitro, sub-micromolar concentrations of PPI inhibited HGC-27 cell proliferation with an IC50 of 0.34 ± 0.06 μM after a 72-h treatment. In vivo, 3 mg/kg PPI significantly inhibited proliferation of HGC-27 tumor cells, with a 78.8% inhibition rate compared to paclitaxel, and demonstrated higher safety. Analysis of MDC and mGFP-LC3 fluorescence, Western blotting and flow cytometry indicated that PPI induced cell cycle arrest in HGC-27 cells by promoting the conversion of LC3-I to LC3-II and by downregulating cyclin B1. Furthermore, Western blotting showed that PPI inhibited the autophagy-regulating PDK1/Akt/mTOR signaling pathway in vitro and in vivo. In addition, immunohistochemistry and TUNEL staining revealed that PPI decreased Ki67 expression and increased the percentage of apoptotic cells in HGC-27 xenograft tumors. These data indicate that PPI is an PDK1/Akt/mTOR signaling inhibitor and of therapeutic relevance for gastric cancer treatment and that the rhizome of Paris polyphylla deserves further clinical investigation as an alternative therapy for gastric cancer.

Programmed death-ligand 1 monoclonal antibody-linked immunoliposomes for synergistic efficacy of miR-130a and oxaliplatin in gastric cancers.

Aim: PD-L1 monoclonal antibody-conjugated miR-130a/oxaliplatin-loaded immunoliposomes were constructed for enhanced therapeutic efficacy against gastric cancer. Materials& methods: The in vitro antitumor efficacy of the immunoliposomes was evaluated by cell viability, cell invasion, cell apoptosis and western blot analysis and in vivo antitumor efficacy was evaluated in a HGC27-bearing tumor xenograft model. Results: The inhibitory role of miR-130a was demonstrated in HGC27 cells by the downregulation of RAB5A and FOCL1 signaling pathways. Consequently, PD-miOXNP exhibited the strongest anticancer activity in vitro compared with any other formulation. PD-miOXNP showed a significantly higher anticancer efficacy in HGC27 tumors with reduced Ki67+ cells and increased TUNEL+ cells for mice group. Conclusion: PD-L1 monoclonal antibody-conjugated immunoliposomes have immense potential to be applied as a next-generation nanomedicine for PD-L1-positive gastric cancers.

Abstract 4022: Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer

Abstract Purpose: N6-methyladenosine (m6A) post-transcriptional RNA modification represents the most abundant epigenetic alteration in eukaryotic cells. These m6A modifications are regulated by a cascade of enzymes and cofactors, categorized as ‘writers’ [methyltransferase-like 3 (METTL3), METTL14, and Wilms’ tumor 1-associating protein (WTAP1)], ‘erasers’ [fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein AlkB homolog 5 (ALKBH5)] and ‘readers’ [YTHDF1, and YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein)]. While emerging evidence indicates that these enzymes play a central role in RNA metabolism (e.g. RNA stability, translation, splicing, transport and localization), their clinical significance, if any, remains unclear. Herein, we for the first time, systematically unraveled the functional role, as well as the clinical significance of m6a regulators in gastric cancer (GC). Experimental design: First, we investigated the expression of the seven m6A regulator genes (FTO, METTL3, METTL14, WTAP, ALKBH5, YTHDF1, and YTHDF2) in a large cohort of 171 GC patients by qRT-PCR assays. Subsequently, a multivariate Cox-regression model was developed using the 7-gene panel to evaluate its predictive potential, followed by Kaplan Meier analyses for survival outcomes. In addition, we undertook a series of functional studies to investigate the oncogenic role of FTO in GC cells, and subsequent validation of our findings in an animal model. Results: Gastric cancer patients with low-expression of METTL3, METTL14, ALKBH5, WTAP and YTHDF1 demonstrated significantly poor OS (p=0.02, 0.001, 0.01, 0.02 and 0.02, respectively), while patients with high FTO expression exhibited markedly worse OS (p&amp;lt;0.0001). Furthermore, the cumulative risk-score derived from these gene panel also significantly associated with poor OS, with a corresponding hazard ratio of 5.47 (95% CI: 3.18-9.41, p&amp;lt;0.0001). We observed that FTO expression was frequently up-regulated in GC cell lines, particularly in those with epithelial-mesenchymal-transition (EMT) features. Functional studies with FTO knockdown in HGC27 and AGS cells inhibited cell proliferation (p=0.005, and &amp;lt;0.0001, respectively) and migratory potential (p&amp;lt;0.0001, respectively); while its overexpression in MKN28 cells resulted in enhanced proliferation and migration (p&amp;lt;0.0001, and 0.007, respectively). Finally, confirming our in-vitro findings, FTO suppression led to significant tumor growth inhibition in a xenograft animal model (p&amp;lt;0.0001). Conclusions: We provide first evidence that m6A regulators may serve as important prognostic biomarkers in GC patients. Our functional studies reveal that FTO is an important oncogene, and may be a novel therapeutic target in patients with gastric cancer. Citation Format: Tadanobu Shimura, Raju Kandimalla, Yuji Toiyama, Yoshinaga Okugawa, Masato Kusunoki, Ajay Goel. Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4022.

Verification of expressions of lncRNA FOXCUT in gastric adenocarcinoma patients and its effects on cell biological function based on TCGA database.

This study aimed to investigate expressions of lncRNA FOXCUT in gastric adenocarcinoma patients and its effects on the cell biological function. Expressions and survival of lncRNA FOXCUT in gastric adenocarcinoma patients (GA) in the Cancer Genome Atlas (TCGA) database were collected. Fifty patients with GA treated in our hospital (patient group) and another 50 contemporaneous normal people (normal group) were collected. Expressions of lncRNA FOXCUT in GES1, SNU-5, HGC-27, SGC-7901, and AGS cells were detected. Also, si-lncRNA FOXCUT and si-NC sequences were transfected to SGC-7901. Si-RNA and si-NC groups were constructed in AGS cells. QRT-PCR was used to detect expressions of lncRNA FOXCUT in samples. MTT, transwell, and flow cytometry were used to detect the proliferation, invasion, and apoptosis of transfected cells. Patients were followed up for 5 years to observe their survival. Expressions of lncRNA FOXCUT in cancer tissues of GA patients in TCGA database were significantly increased (p<0.001). The survival rate of patients with low expressions of lncRNA FOXCUT was significantly increased (p=0.017, p=0.047). LncRNA FOXCUT is closely related to patients' tumor diameter, lymph node metastasis, TNM staging, and differentiation degree (p<0.05). LncRNA FOXCUT has high clinical value in disease diagnosis. Multivariate Cox regression analysis found that tumor diameter, lymph node metastasis, and lncRNA FOXCUT were independent prognostic factors. Compared with GES1, expressions of lncRNA FOXCUT in GA cells increased significantly (p<0.05), the proliferation and invasion ability of si-RNA group decreased significantly (p<0.05) compared with si-NC group, and the apoptosis rate of si-RNA group was significantly lower than that of si-NC group (p<0.05). We showed that the inhibition of the expressions of lncRNA FOXCUT can reduce the proliferation and invasion of GA cells and increase apoptosis, which can be used as a potential therapeutic target for GA.

Abstract 4314: Reduced m6A modification predicts malignant phenotypes and augmented tumorigenic signaling in gastric cancer

Abstract Purposes: As the most abundant epigenetic modification on mRNAs and long non-coding RNAs, N6-methyladenosine (m6A) extensively exists in mammalian cells. Controlled by writers (methyltransferases), readers (signal transducers) and erasers (demethylases), m6A influences mRNA structure, maturation and stability, thus regulating protein expression in a post-transcriptional manner. Nevertheless, current understandings to m6A’s pathological roles in tumorigenesis, especially in gastric cancer (GC) remain to be unveiled. In this study, by incorporating multiple datasets and in vitro experiments, we assessed m6A’s clinical-pathological relevance to GC and explored the underlying mechanisms. Methods: The protein and mRNA expressions of canonical m6A regulators (METTL3/METTL14 as writers, YTHDF1/YTHDF2/YTHDF3 as readers and ALKBH5/FTO as erasers) were merged as signatures to indicate levels of m6A modification. Changes of clinical variables, mutations and molecules were assessed in patients with different m6A indications. Pathway and phenotype changes predicted to be affected by m6A were assessed in vitro with gastric cancer HGC27 and MG803 cell lines. Results: We discovered that writers and readers were potential tumor suppressive, while erasers were potential oncogenic factors in GC. Low-m6A modification was an indication for adverse clinical outcomes and was correlated with specific cancer driving mutations, including CDH1, AR and GLI3. Oncogenic signaling and phenotypes were selectively enriched in m6A groups. Through in vitro experiments, we proved that reduced m6A (represented by METTL14 knockdown) promoted GC proliferation and invasiveness potentially through activating Wnt and PI3K-Akt signaling, while restored m6A (represented by FTO knockdown) reversed these phenotypical and molecular changes. Conclusions: Our work demonstrated that m6A is a potential prognostic predictor in gastric cancer and further reveals the reduction of m6A activates oncogenic signaling and promotes gastric cancer malignancy. Citation Format: Cheng Zhang, Mengqi Zhang, Sai Ge, Xiaotin Lin, Wenwen Huang, Ying Gan, Jing Gao, Lin Shen. Reduced m6A modification predicts malignant phenotypes and augmented tumorigenic signaling in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4314.