Autophagy In Gastric Cancer Cells Research Articles

Exploring the Mechanism of KLF15 on the Biological Activity and Autophagy of Gastric Cancer Cells based on PI3K/Akt/Mtor Signaling Pathway.

To explore the mechanism of KLF15 on the biological activity and autophagy of gastric cancer cells based on the PI3K/Akt/mTOR signaling pathway. The gastric cancer AGS cells were divided into the Con group, pcDNANC group, pcDNA-KLF15 group, LY294002 group and IGF-1 group. RT-PCR was used to detect the expression of KLF15 in human gastric mucosal cells and gastric cancer cells; MTT method to detect cell proliferation; Transwell method to detect cell invasion; flow cytometry to detect cell apoptosis; Western blotting to detect PI3K, Akt, mTOR in cells, LC3, Beclin1, p62 protein expression.P<0.05 was used to indicate statistical significance. Compared with the human gastric mucosal cell line GES-1 cells, the expression of KLF15 in human gastric cancer cell lines MKN-28, MFC, SCG-7901 and AGS cells was significantly decreased, And the expression of KLF15 in AGS cells, was the lowest (P=0.006). Compared with the Con group, The expression of KLF15 in the cells of the PCDNA-KLF15 group was significantly increased (P=0.018); There was no significant difference in the expression of KLF15 between the Con group and the PCDNA-NC group (P=0.225). Compared with the Con group, the proliferation and invasion abilities of the cells in the pcDNA-KLF15 group were significantly reduced, And the apoptosis ability was significantly increased (P=0.019). The ratio of LC3II/LC31 and the expression of Beclin1 Protein in the control group were significantly higher than those in the Con group (P=0.017). Overexpression of KLF15 can inhibit the proliferation and invasion of Gastric cancer cells and promote cell apoptosis and autophagy, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

RACGAP1 drives proliferation, migration and invasion and suppresses autophagy of gastric cancer cells via inhibiting SIRT1/Mfn2.

Gastric cancer is the most frequent gastrointestinal malignancy with a poor prognosis. Rac GTPase activation protein 1 (RACGAP1) is a novel tumor promotor, whose detailed effect on gastric cancer remains to be further elucidated. Hence, this study identifies the action of RACGAP1 on gastric cancer and investigates the potential mechanism. RACGAP1 expression in gastric cancer was analyzed based on the data of The Cancer Genome Atlas (TCGA) database. Cell proliferation was measured by CCK-8 and colony formation assay. Cell migration and invasion were evaluated by transwell assay. Cell apoptosis was assessed by flow cytometry. Cell autophagy was evaluated via determining LC3. RACGAP1 presented at high level in gastric cancer cells. Overexpressed RACGAP1 potentiated gastric cancer cell proliferation, migration, and invasion. Besides, silenced RACGAP1 induced cell apoptosis and autophagy. Furthermore, RACGAP1 suppressed the expression of SIRT1 and Mfn2. RACGAP1 was overexpressed in gastric cancer. RACGAP1 potentiated aggressive behaviors of gastric cancer, and suppressed cell apoptosis and autophagy via modulating SIRT1/Mfn2. RACGAP1 may be a valuable target in the treatment of gastric cancer.

ABBV-744 induces autophagy in gastric cancer cells by regulating PI3K/AKT/mTOR/p70S6k and MAPK signaling pathways

The mortality rates of gastric cancer remain high due to limited therapeutic strategies. As a highly selective inhibitor of the BD2 domain of BET family proteins, ABBV-744 has potent chemotherapeutic activity against various human solid tumors. However, whether ABBV-744 has potential anti-tumor effects in gastric cancer remain largely unknown. In this study, we evaluated the effect of ABBV-744 on gastric cancer cells and explored the possible underlying mechanisms. We found that ABBV-744 inhibited the growth of gastric cancer cells and patient-derived tumor organoids in a dose-dependent manner. Cellular experiments revealed that ABBV-744 induced mitochondria damage, reactive oxygen species accumulation, cell cycle arrest and apoptotic cell death in gastric cancer cells. Transcriptomic analysis using RNA-sequencing data identified autophagy as a crucial pathway involved in the cell death caused by ABBV-744. Mechanically, further studies showed that ABBV-744 induced autophagy flux in gastric cancer cells by inactivating PI3K/AKT/mTOR/p70S6k and activating the MAPK signaling pathways. In vivo mouse xenograft studies demonstrated that ABBV-744 significantly suppressed the growth of gastric cancer cells via inducing autophagy. Taken together, our results suggest that ABBV-744 is a novel drug candidate for gastric cancer.

CircRNA_15430 reduced by Helicobacter pylori infection and suppressed gastric cancer progression via miR-382-5p/ZCCHC14 axis

BackgroundHelicobacter pylori (H.pylori, HP) is one of the main causes of gastric cancer (GC). CircRNAs have been reported to play a crucial role in developing many types of cancer. However, the role of circRNAs in the development and progression of HP infected-GC has not been studied.MethodsThe location of circRNA_15430 in GC cells were detected by nuclear and cytoplasmic RNA fractionation and RNA fluorescence in situ hybridization analysis (FISH) assays, and circRNA_15430, miR-382-5p and ZCCHC14 expression in GC cell lines and tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The function of circRNA_15430 in GC cells were examined by using colony formation, cell counting kit-8 (CCK-8) and Transwell assays, flow cytometry and laser scanning confocal microscopy. The protein levels were detected by Western blotting. Whether circRNA_15430 sponges miR-382-5p was monitored with a dual-luciferase reporter assay. Furthermore, circRNA_15430 was analyzed in vivo in tumor growth with nude mice.ResultsCircRNA_15430 is primarily localized in the cytoplasm of GC cells, and downregulated in the GC cell lines and tissues, and is negatively correlated with the tumor size. Downregulation of circRNA_15430 promotes proliferation, migration and suppresses cell apoptosis and autophagy in GC cells. Mechanically, circRNA_15430 acts as a miR-382-5p sponge, alleviating the inhibitory effect of miR-382-5p on its target ZCCHC14. Knockdown circRNA_15430 enhances tumor growth in vivo. In addition, circRNA_15430 was reduced in HP + gastritis tissues and HP-infected MGC-803 cells, reversing the pro-HP effect on autophagy. Additionally, miR-382-5p was increased in HP + gastritis tissue and HP-infected MGC-803 cells while ZCCHC14 decreased in HP-infected MGC-803 cells. MiR-382-5p reverses the effect of si-ZCCHC14 on autophagosome numbers in MGC-803 cells.ConclusionsTherefore, circRNA_15430 plays an inhibitory role in GC and regulates the progression of HP infection-related GC, providing a novel molecular marker for GC therapy.

Thymoquinone induces apoptosis and protective autophagy in gastric cancer cells by inhibiting the PI3K/Akt/mTOR pathway.

Gastric cancer (GC) is often diagnosed in the advanced stages with a poor prognosis. Thymoquinone (TQ) is known for its antitumor activity; however, the specific mechanism in GC remains unknown. In our study, TQ inhibited GC cell proliferation and induced apoptosis and autophagy in a concentration-dependent manner. Transmission electron microscopy showed increased autophagosome formation in GC cells treated with TQ. Meanwhile, the LC3B puncta and LC3BII protein levels were significantly increased in GC cells, while p62 expression was significantly decreased. The autophagy inhibitor, Bafilomycin A1 enhanced TQ-inhibited proliferation and TQ-induced apoptosis, suggesting that TQ-induced autophagy has a protective effect on GC cells. Furthermore, TQ decreased the phosphorylation levels of phosphatidylinositol-4,5-bisphosphate 3 kinase (PI3K), protein kinase B (Akt), and mechanistic target of rapamycin (mTOR). The PI3K agonist partially rescued TQ-induced autophagy and apoptosis. Finally, in vivo experiments showed that TQ could inhibit tumor growth and promote apoptosis and autophagy. This study provides new insights into the specific mechanism for the anti-GC effect of TQ. TQ inhibits the proliferation of GC cells and induces apoptosis and protective autophagy by inhibiting the PI3K/Akt/mTOR pathway. The results suggest that the combination of TQ and autophagy inhibitors might be a potential chemotherapeutic strategy for GC.

SIRT1 mediated gastric cancer progression under glucose deprivation through the FoxO1-Rab7-autophagy axis.

Silent mating type information regulator 2 homolog 1 (SIRT1) and autophagy have a two-way action (promoting cell death or survival) on the progression and treatment of gastric cancer (GC) under different conditions or environments. This study aimed to investigate the effects and underlying mechanism of SIRT1 on autophagy and the malignant biological behavior of GC cells under conditions of glucose deprivation (GD). Human immortalized gastric mucosal cell GES-1 and GC cell lines SGC-7901, BGC-823, MKN-45 and MKN-28 were utilized. A sugar-free or low-sugar (glucose concentration, 2.5 mmol/L) DMEM medium was used to simulate GD. Additionally, CCK8, colony formation, scratches, transwell, siRNA interference, mRFP-GFP-LC3 adenovirus infection, flow cytometry and western blot assays were performed to investigate the role of SIRT1 in autophagy and malignant biological behaviors (proliferation, migration, invasion, apoptosis and cell cycle) of GC under GD and the underlying mechanism. SGC-7901 cells had the longest tolerance time to GD culture conditions, which had the highest expression of SIRT1 protein and the level of basal autophagy. With the extension of GD time, the autophagy activity in SGC-7901 cells also increased. Under GD conditions, we found a close relationship between SIRT1, FoxO1 and Rab7 in SGC-7901 cells. SIRT1 regulated the activity of FoxO1 and upregulated the expression of Rab7 through deacetylation, which ultimately affected autophagy in GC cells. In addition, changing the expression of FoxO1 provided feedback on the expression of SIRT1 in the cell. Reducing SIRT1, FoxO1 or Rab7 expression significantly inhibited the autophagy levels of GC cells under GD conditions, decreased the tolerance of GC cells to GD, enhanced the inhibition of GD in GC cell proliferation, migration and invasion and increased apoptosis induced by GD. The SIRT1-FoxO1-Rab7 pathway is crucial for the autophagy and malignant biological behaviors of GC cells under GD conditions, which could be a new target for the treatment of GC.

NRP1 regulates autophagy and proliferation of gastric cancer through Wnt/β-catenin signaling pathway.

Gastric cancer possesses high lethality rate, and its complex molecular mechanisms of pathogenesis lead to irrational treatment outcomes. Autophagy plays a dual role in cancer by both promoting and suppressing the cancer. However, the role of autophagy in gastric cancer is still vague. Therefore, in this study, we first obtained autophagy-related genes from the Human Autophagy Database, and then applied consensus clustering analysis to analyse the molecular subtypes of gastric cancer samples in the TCGA database. The genes obtained after subtyping were then applied to construct risk prognostic model. Following this, PCA and tSNE assessed risk scores with good discriminatory ability for gastric cancer samples. The results of Cox regression analysis and time-dependent ROC curve analysis indicated that the model had good risk prediction ability. Finally, NRP1 was selected as the final study subject in the context of expression pairwise analysis, Kaplan-Meier curves and external validation of the GEO dataset. In vitro experiments showed that NRP1 has the ability to regulate the proliferation and autophagy of gastric cancer cells by affecting the Wnt/β-catenin signalling pathway. Similarly, in vivo experiments have shown that NRP1 can affect tumour growth in vivo. We therefore propose that NRP1 can be used as both a prognostic factor and a therapeutic target through the regulation of autophagy in gastric cancer.

Inactivation of ZSCAN18 by promoter hypermethylation drives the proliferation via attenuating TP53INP2-mediated autophagy in gastric cancer cells

BackgroundZinc finger and scan domain containing 18 (ZSCAN18) belongs to the zinc finger transcription factor superfamily, which consists of hundreds of members that play critical roles in all steps of tumorigenesis.MethodsThis study aims to investigate the roles of ZSCAN18 in gastric cancer (GC). The expression level in GC and the clinicopathologic features of ZSCAN18 were detected by immunohistochemistry staining. Methylation of ZSCAN18 promoter in GC tissues and cell lines was analyzed via MassARRAY; the same method was used to detect GC cell lines demethylated by 5-aza-2′-deoxycytidine treatment. The biological function of ZSCAN18 in GC cells was verified by in vitro and in vivo experiments. The downstream molecular mechanism of ZSCAN18 was explored using RNA next-generation sequencing, immunofluorescence and chromatin immunoprecipitation.ResultsOur work revealed ZSCAN18 expression was markedly reduced in GC tissues compared with adjacent normal tissues as a result of hypermethylation in GC. Likewise, ZSCAN18 expression was significantly reduced in a panel of GC cell lines as a result of the densely methylated ZSCAN18 promoter. Functionally, ZSCAN18 overexpression inhibited the biological progression of GC cells, which was characterized by weaken proliferation, enhanced autophagy and suppressed tumor growth. ZSCAN18 acted as a transcription factor and played an important role in binding to the promoter of tumor protein 53-induced nuclear protein 2 (TP53INP2), and we also confirmed the anti-tumor effect of TP53INP2 in GC. Furthermore, the knockdown of TP53INP2 alleviated the inhibiting effects of ZSCAN18 in GC cells by in vitro and in vivo experiments.ConclusionsCollectively, this study unveiled that ZSCAN18 played an anticancer role in GC by promoting autophagy and transcriptional regulation of TP53INP2 and provided a promising target for the diagnosis and treatment of GC.

Low Expression of GIGYF1 Inhibits Metastasis, Proliferation, and Promotes Apoptosis and Autophagy of Gastric Cancer Cells.

GRB10 interacting GYF protein 1 (GIGYF1) binds to the N-terminal region of Grb10, regulates multiple signaling pathways. However, it is not clear what happens to cell proliferation, metastasis, apoptosis, and autophagy when the expression level of GIGYF1 gene is reduced. Detection of GIGYF1 expression in clinical tissue specimens and gastric cancer (GC) cell lines by quantitative Real-time PCR (qRT-PCR), GIGYF1 gene was knocked down in MGC-803 cells using small interfering RNA, the effect of GIGYF1 gene on cell metastasis was detected using Transwell assay and wound healing assay, the effect on cell proliferation was detected using plate cloning assay and cck-8 assay, the effect on apoptosis was detected using flow cytometry, autophagosomes were detected using laser confocal microscopy, and the effect on protein expression was detected using immunofluorescence and Western blotting. GIGYF1 gene expression was higher in tumor tissue samples than in paracancer tissue samples, and higher in human GC cell lines than in human normal gastric epithelial cells. GIGYF1 gene knockdown inhibited cell migration, scratch healing ability and EMT process, weakened cell proliferation ability, increased apoptosis rate, promoted the formation of autophagosomes, and changed the corresponding protein expression level. Meanwhile, GIGYF1 knockdowns inhibited the ERK and AKT signaling. In conclusion, the low expression of GIGYF1 gene can inhibit the occurrence and progression of gastric cancer, during which the ERK and AKT signaling pathways are inhibited.

Role of IRE1 in autophagy induced by vitamin E succinate in human gastric cancer cell

To investigate the role of inositol-requiring enzyme 1(IRE1) in autophagy of human gastric cancer cells induced by vitamin E succinate(VES). Human gastric cancer SGC-7901 cells were cultured in vitro and divided into solvent control group(0.1% ethanol absolute), different doses(5, 10, 15 and 20 μg/mL) VES group, 4μ8C group, and VES + 4μ8C group. The endoplasmic reticulum stress-related molecules glucose regulated protein 78(GRP78) and C/EBP homologous protein(CHOP), autophagy marker microtubule associated Protein1 light chain 3(LC3), Beclin-1, unfolded protein response branching pathway Inositol-requiring enzyme 1(IRE1), X box-binding protein 1(XBP1), c-Jun n-terminal kinase(JNK) and p-JNK were detected by Western blot in the solvent control group and different doses of VES group. IRE1 was inhibited by 4μ8C. The expressions of IRE1, XBP1, JNK, p-JNK, GRP78 and CHOP were detected by Western blot, and the expressions of LC3 and Beclin-1 were detected. The expression of GRP78(1.16±0.06) and CHOP(1.36±0.11) in 20 μg/mL VES group were significantly higher than those in solvent control group GRP78(0.36±0.10) and CHOP(0.48±0.05)(P&lt;0.001). The expression of Beclin-1(1.09±0.20) and LC3-Ⅱ/LC3-Ⅰ(1.29±0.03) in 20 μg/mL VES group were significantly higher than those in solvent control group(0.27±0.07) and LC3-Ⅱ/LC3-Ⅰ(0.43±0.06)(P&lt;0.001). The expression levels of IRE1(1.07±0.20), XBP1(1.33±0.07) and p-JNK/JNK(1.19±0.31) in 20 μg/mL VES group were significantly higher than those in the solvent control group(P&lt;0.01). After IRE1 is inhibited: The expression level of IRE1(0.63±0.27), XBP1(0.74±0.09), p-JNK/JNK(0.35±0.04), GRP78(0.66±0.02), CHOP(0.51±0.02), LC3-Ⅱ/LC3-Ⅰ(0.72±0.01), Beclin-1(0.70±0.15) was significantly lower than that of VES group(P&lt;0.05). VES may participate in the regulation of autophagy in gastric cancer cells by upregulating IRE1 pathway.

20(S)-Protopanaxadiol from Panax ginseng Induces Apoptosis and Autophagy in Gastric Cancer Cells by Inhibiting Src.

20(S)-protopanaxadiol (PPD), a metabolite of Panax ginseng, has multiple pharmacological properties. However, the effects of PPD against human gastric cancer have not been elucidated. Our purpose in this study was to investigate if PPD has anticancer effects against human gastric cancer in vitro. Cell viability, migration, clone formation, and invasion were assessed to explore the effects of PPD on cancer cells. PI and annexin V staining as well as immunoblotting were employed to determine if PPD-induced apoptosis and autophagy of MKN1 and MKN45 cells. The target of PPD was identified using immunoblotting, overexpression analysis, and flow cytometric analysis. PPD exhibited significantly suppressed cell viability, migration, colony formation, and invasion. Phosphorylation of Src and its down-stream effectors were inhibited by PPD.PPD-enhanced apoptosis and autophagy in a dose- and time-dependent manner by inhibiting Src. Collectively, our results demonstrate that PPD induces apoptosis and autophagy in gastric cancer cells in vitro by inhibiting Src.

Exosome-mediated transfer of circ_0063526 enhances cisplatin resistance in gastric cancer cells via regulating miR-449a/SHMT2 axis.

Exosomes are critical mediators of intercellular communication. Exosomal circular RNAs (circRNAs) have been reported to play critical roles in the development of chemoresistance in various tumors, including gastric cancer. However, the role of exosomal circ_0063526 in cisplatin (CDDP) resistance of gastric cancer is still unclear. The expression of circ_0063526, microRNA-449a (miR-449a) and serine hydroxymethyltransferase 2 (SHMT2) mRNA was determined by quantitative real-time PCR (qRT-PCR). Cell viability was assessed by the Cell Counting Kit-8 assay. Cell migration and invasion were evaluated by the transwell assay and wound healing assay. Western blot assay was used to measure the protein expression of light Chain 3 (LC3) I/II, p62 and SHMT2. Exosomes were detected using transmission electron microscopy. The size distribution of exosomes was analyzed by nanoparticle tracking analysis. The interaction between miR-449a and circ_0063526 or SHMT2 was confirmed by a dual-luciferase reporter and RNA pull-down assays. Circ_0063526 expression was increased in gastric cancer tissues and cells and CDDP-resistant cells. Extracellular circ_0063526 could be packaged into exosomes and transmitted to sensitive cells, thus disseminating CDDP resistance. Knockdown of exosomal circ_0063526 inhibited CDDP resistance via suppressing migration, invasion and autophagy in gastric cancer cells. Moreover, circ_0063526 was identified as a molecular sponge of miR-449a to upregulate SHMT2 expression. Further, exosomal circ_0063526 regulated SHMT2 expression to enhance CDDP resistance of gastric cancer cells. Additionally, high expression of exosomal circ_0063526 in serum was associated with poor response to CDDP treatment in gastric cancer patients. Exosomal circ_0063526 facilitated CDDP resistance in gastric cancer via regulating the miR-449a/SHMT2 axis.

Nintedanib Induces the Autophagy-Dependent Death of Gastric Cancer Cells by Inhibiting the STAT3/Beclin1 Pathway.

Tyrosine kinase inhibitors are currently the most widely studied targeted therapies for gastric cancer. As a triple tyrosine inhibitor, nintedanib can alleviate the progression of a variety of cancers, but it is poorly studied in gastric cancer. To investigate the effect of nintedanib on gastric cancer. This study investigated nintedanib's effect on gastric cancer autophagy in vivo and in vitro, and the activity and morphological changes of gastric cancer cells were detected by MTT and HE staining. Proliferation, migration, invasion, and EMT-related marker proteins of AGS and MKN-28 cells were detected. The effects of nintedanib on autophagy in gastric cancer cells were detected by acridine orange, immunofluorescence, and Western blotting assays. The regulation of nintedanib on STAT3 and Beclin1 was detected by qPCR and Western blotting assays. Subsequently, the effects of nintedanib on the tumor STAT3/Beclin1 pathway were verified by stably overexpressing STAT3 in gastric cancer cell lines and tumor-bearing experiments in nude mice. The results showed that nintedanib could inhibit gastric cancer cells' proliferation and EMT process. Meanwhile, autophagy was induced in AGS and MKN-28 cells, and the expression of autophagy-related protein Beclin1 was upregulated, and the phosphorylation level of STAT3 was downregulated. Nintedanib inhibited STAT3 phosphorylation and upregulated Beclin1 to inhibit tumor growth in gastric cancer cell lines with stable STAT3 overexpression and tumor-bearing experiments in nude mice. By inhibiting STAT3, nintedanib upregulated Beclin1 and caused autophagic death in gastric cancer cells.

Interleukin-8 Regulates the Autophagy and Apoptosis in Gastric Cancer Cells via Regulating PI3K/Akt Signaling Pathway.

Objective To explore the role and mechanism of interleukin-8-mediated autophagy regulation of gastric cancer (GC) cells in GC. Methods After cell culture, the SGC7901 cell line was separated into the control group and IL-8 (20 ng/mL) group, IL-8 (40 ng/mL) group, and IL-8 (60 ng/mL) group, to verify the effects of the PI3K/Akt signal path on the modulation of autophagy in GC cells. Western blot detected autophagy markers, ATG12-ATG5 complexes, autophagy-associated pathways, and apoptosis-associated factors in GC cells. Transwell was utilized to identify invasion capability. Results Compared with the control group, the expression of LC3II, Atg5, ATG7, Beclin1, Bax, C-cas3, C-cas9, P-PI3K, P-Akt, and ATG12-ATG5 was remarkably elevated in the IL-8 (60 ng/mL) group, IL-8 (20 ng/mL) group, and the IL-8 (40 ng/mL) group. The expression of P62 and Bcl-2 in the IL-8 (60 ng/mL) group was also lower than that of the IL-8 (20 ng/mL) group and IL-8 (40 ng/mL) group, in contrast to the controls. The invasive quantity of GC SGC7901 cells in the IL-8 (60 ng/mL) group was also remarkably higher in contrast to the IL-8 (20 ng/mL) and IL-8 (40 ng/mL) groups. The relative expressions of LC3II, Atg5, ATG7, Beclin1, Bax, C-cas3, C-cas9, P-PI3K, P-Akt, and ATG12-ATG5 complex proteins in LY294002 group were considerably elevated. LC3II, Atg5, ATG7, Beclin1, Bax, C-cas3, C-cas9, P-PI3K, P-Akt, and ATG12-ATG5 were decreased in the IL-8 + LY294002 group. The relative expressions of P62 and Bcl-2 proteins in the IL-8 + LY294002 group were remarkably elevated, and the invasion of SGC7901 cells in the IL-8 group was elevated. In contrast to the IL-8 group, the invasion quantity of gastric cancer SGC7901 cells in the IL-8 + LY294002 group was considerably decreased. Conclusion IL-8 promotes autophagy and aggression and suppresses apoptosis of GC SGC7901 cells by regulating PI3K/AKT pathway phosphorylation.

APY0201 Represses Tumor Growth through Inhibiting Autophagy in Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common cancers globally. There are currently few effective chemotherapeutic drugs available for GC patients. The inhibitors of phosphatidylinositol kinase containing an FYVE finger structure (PIKfyve) have shown significant anticancer effects in several types of cancers, but their effectiveness in GC remains unknown. In this study, we investigate the effect of APY0201, an inhibitor of PIKfyve, on GC tumor growth and its mechanism of action. It was found that APY0201 inhibited GC cell proliferation in in vitro GC cell model, organoid model, and in vivo xenograft tumor model. Through analyzing cell autophagy, we found that APY0201 might block autophagic flux by impairing lysosome degradation function of GC cells, inducing the accumulation of autophagosomes, thus causing the inhibition of GC cell proliferation. We also found that APY0201 induced G1/S phase arrest in GC cells. Importantly, APY0201 was also effective in inducing repression of autophagy and cell cycle arrest in the mouse tumor xenograft. Our results suggest that APY0201 could be a new promising therapeutic option for GC.

The Overexpression of TOB1 Induces Autophagy in Gastric Cancer Cells by Secreting Exosomes

We previously confirmed that transducer of ERBB2, 1 (TOB1) gene, can induce autophagy in gastric cancer cells. Studies have shown the biogenesis of exosomes overlaps with different autophagy processes, which helps to maintain the self-renewal and homeostasis of body cells. This study is aimed at verifying whether overexpressing TOB1 induces autophagy by secreting exosomes in gastric cancer cells and its underlying mechanisms. Differential ultracentrifugation was used to extracted the exosomes from the culture medium of gastric cancer cell line AGS-TOB1 ectopically overexpressing TOB1 (exo-AGS-TOB1, experimental group) and AGS-empty-vector cell line with low expression of endogenous TOB1 (exo-AGS-Vector, control group). Exosomal markers CD9 and TSG101 were determined in both the cell supernatants of exo-AGS-TOB1 and exo-AGS-Vector by Western blot. Under the transmission electron microscope (TEM), the exosomes were round and saucer-like vesicles with double-layer membrane structure, and the vesicles showed different translucency due to different contents. The peak size of exosomes detected by nanoparticle tracking analysis (NTA) was about 100 nm. When the exosomes of exo-AGS-TOB1 and exo-AGS-Vector were cocultured with TOB1 knockdown gastric cancer cell line HGC-27-TOB1-6E12 for 48 hours, the conversion of autophagy-related protein LC3-I to LC3-II in HGC-27-TOB1-6E12 gastric cancer cells cocultured with exo-AGS-TOB1 was significantly higher than that in the control group, and the ratio of LC3-II/LC3-I was statistically different (P < 0.05). More autophagosomes in HGC-27-TOB1-6E12 cells cocultured with exo-AGS-TOB1 for 48 hours were observed under TEM, while fewer autophagosomes were found in the control group. Lastly, miRNAs were differentially expressed by cell supernatant-exosomal whole transcriptome sequencing. Thus, our results provide new insights into TOB1-induced autophagy in gastric cancer.

Suppression of JNK/ERK dependent autophagy enhances Jaspine B derivative-induced gastric cancer cell death via attenuation of p62/Keap1/Nrf2 pathways

Gastric cancer is one of the most common cancers with few effective treatments, a new treatment agent is desperately needed. C-2, a Jaspine B derivative, has shown anti-cancer efficacy in gastric cancer cells. The anti-cancer mechanism, however, remains unknown. As a result, we investigate the anti-cancer effect and the underlying mechanism of C-2 in gastric cancer cells. The results showed that C-2 selectively reduced the proliferation of gastric cancer cells when compared to normal epithelial gastric cells. Western blotting and flow cytometry further demonstrated that Caspase9 is involved in causing cell death. Meanwhile, C-2 triggered autophagy in gastric cancer cells, inhibition of which with LY294002 can enhance the anti-proliferative activity of C-2. Next, we found that C-2 triggered autophagy through activating JNK/ERK, and that inhibitors of these proteins exacerbated C-2 induced cell death. Mechanically, enhanced phosphorylation of JNK/ERK elevated Beclin-1 by disturbing Beclin-1/Bcl-xL or Beclin-1/Bcl-2 complexes, resulting in autophagy and up-regulation of p62. Finally, p62 binds Keap1 competitively to release Nrf2, boosting Nrf2 translocation from the cytoplasm to the nucleus and triggering expression of Nrf2 target genes, so enhancing survival. C-2 inhibited the growth of gastric cancer cells, while JNK/ERK dependent autophagy antagonized C-2 induced cell growth inhibition through p62/Keap1/Nrf2 pathway.

Narciclasine induces autophagy-mediated apoptosis in gastric cancer cells through the Akt/mTOR signaling pathway

BackgroundGastric cancer is a common gastrointestinal cancer and currently has the third-highest mortality rate. Research shows that the natural compound narciclasine has a variety of biological activities. The present study aimed to investigate the effect of narciclasine on gastric cancer cells and its molecular mechanisms and determine whether this compound could be a novel therapy for gastric cancer.MethodsMTT and clone assays were employed to detect the proliferation of gastric cancer cells. The cell apoptosis was detected by flow cytometry. The formation of autophagosomes and autophagosomal lysosomes was observed by transmission electron microscopy and laser confocal scanning microscopy. Western blotting was used to detect the expression of apoptosis, autophagy and Akt/mTOR pathway-related proteins.ResultsIn this study, we found that narciclasine could inhibit the proliferation of gastric cancer cells and promote apoptosis in gastric cancer cells. Further experiments showed that narciclasine promoted the levels of autophagy proteins LC3-II, Atg-5 and Beclin-1, reduced the expression of the autophagy transporter p62, and increased autophagic flux. By using the autophagy inhibitors 3-MA and CQ, it was shown that narciclasine could induce autophagy-mediated apoptosis in gastric cancer cells. Finally, we found that narciclasine had no significant effects on the total content of Akt and mTOR in gastric cancer cells, and it involved autophagy in gastric cancer cells by reducing the phosphorylation level of p-Akt and p-mTOR.ConclusionsNarciclasine can induce autophagy-dependent apoptosis in gastric cancer cells by inhibiting the phosphorylation level of Akt/mTOR and thus reduce the proliferation of gastric cancer cells.

LncRNA CCAT1 Upregulates ATG5 to Enhance Autophagy and Promote Gastric Cancer Development by Absorbing miR-140-3p.

Long noncoding RNA colon cancer-associated transcript 1 (LncRNA CCAT1) is highly expressed in gastric cancer tissues and plays a role in autophagy. However, the underlying mechanism still needs to be further clarified. To study the role of LncRNA CCAT1 in regulating autophagy of gastric cancer cells, analyze its downstream targets, and elucidate the mechanism. qPCR detected the expression of LncRNA CCAT1 in gastric cancer cells. The proliferation, migration, and invasion ability of LncRNA CCAT1 and the expression level of autophagy-related proteins in gastric cancer cells were detected. Bioinformatics method predicted the downstream targets of LncRNA CCAT1, and they were verified by dual-luciferase assay. The relationship between LncRNA CCAT1, miR-140, and ATG5 was verified by co-transfection, and the expression levels of ATG5 and ATG5-ATG12 complex proteins were detected. Finally, the role of LncRNA CCAT1 in vivo was confirmed by gastric cancer transplantation model. LncRNA CCAT1 was highly expressed in gastric cancer cells. LncRNA CCAT1 can promote the proliferation, migration, invasion, and autophagy activity of gastric cancer cells. LncRNA CCAT1 can bind to miR-140-3p and regulate its expression, while miR-140-3p further regulates the expression of ATG5. Overexpression of LncRNA CCAT1 can promote tumor growth in nude mice. After LncRNA CCAT1 silencing, the positive expression rate of ATG5 in nude mice was low. LncRNA CCAT1 may inhibit the expression of miR-140-3p by sponge adsorption, thus weakening its inhibitory effect on ATG5. Eventually, gastric cancer cells were more prone to autophagy under the pressure of stress.

Effect and mechanism of action of SLP-2 on the apoptosis and autophagy of gastric cancer cells.

This study was designed to investigate the effect of stomatin-like protein 2 (SLP-2) on the apoptosis and autophagy of gastric cancer cells and its underlying mechanism. The expression of SLP-2 was detected in human gastric cancer cell lines (AGS, MKN-45 and NCI-N87) and a human gastric epithelial cell line (GES-1) using reverse transcription-quantitative PCR and western blot analysis. SLP-2-specific small interfering RNA (siRNA) was transfected into NCI-N87 cells. Cell Counting Kit-8 was used to detect cell proliferation. Apoptosis rates were measured using flow cytometry. Autophagosomes were observed by transmission electron microscopy. The expression levels of Annexin A2 (ANXA2), β-catenin, Bcl-2, Bax, Beclin-1 and LC3-II/I were also measured. The results demonstrated that SLP-2 siRNA transfection significantly reduced cell proliferation and increased cell apoptosis. The mitochondria were severely damaged, and a large number of autophagosomes were seen in SLP-2 siRNA-transfected NCI-N87 cells. Furthermore, the expression levels of ANXA2, β-catenin and Bcl-2 were downregulated, whereas those of Bax, Beclin-1 and LC3-II/I were upregulated following SLP-2 siRNA transfection. In conclusion, SLP-2 silencing can inhibit proliferation and induce apoptosis and autophagy of gastric cancer cells, and this effect may be related to the inhibition of ANXA2/β-catenin signaling pathway.