Human Gastric Cells Research Articles

High KHSRP expression promotes gastric adenocarcinoma metastasis: the mediating role of the JAK1/STAT3 signaling axis

To investigate the regulatory effect of KHSRP on progression of gastric adenocarcinoma and the role of the JAK1/STAT3 signaling axis in mediating its effect. KHSRP mRNA expression level was detected using qRT-PCR in 120 pairs of gastric adenocarcinoma and adjacent tissues, 4 gastric adenocarcinoma cell lines (MKN-28, HGC-27, CRL-5822, and SNU-1) and normal human gastric mucosal GES-1 cells. In HGC-27 cells with KHSRP knockdown and SNU-1 cells with KHSRP overexpression, cell proliferation, migration, invasion and expression levels of JAK/STAT were evaluated using CCK-8 assay, Transwell migration and invasion assays, and Western blotting. In BALB/c-nude mice, HGC-27 cells with KHSRP knockdown and SNU-1 cells overexpressing KHSRP were injected either subcutaneous or via the tail vein to observe subcutaneous xenograft growth and lung metastasis of the tumor cells. Gastric adenocarcinoma tissues and cell lines all showed significantly increased KHSRP expression as compared with the adjacent tissues and GES-1 cells. In HGC-27 cells, KHSRP knockdown significantly inhibited cell proliferation, migration and invasion, while KHSRP overexpression enhanced the malignant behaviors of SNU-1 cells. In nude mice, inoculation of HGC-27 cells with KHSRP knockdown resulted in smaller tumor volume and weight, slower cell proliferation rate and fewer lung metastatic foci, and KHSRP-overexpressing SNU-1 cells produced the opposite results. KHSRP knockdown in HGC-27 cells significantly down-regulated the expression levels of JAK1 and STAT3, which were obviously increased in KHSRP-overexpressing SNU-1 cells. High expressions of KHSRP promote progression and metastasis of gastric adenocarcinoma possibly by regulating the JAK1/STAT3 signaling axis.

ABRACL upregulated by transcription factor CBX4 promotes proliferation and migration and inhibits the apoptosis of gastric cancer cells.

Gastric cancer (GC) is a predominant health concern in many countries. Actin-binding Rho activating C-terminal-like (ABRACL) belongs to a new family of low molecular weight proteins and has been implicated in cancers. This study was implemented to elucidate the role and mechanism of ABRACL in GC. mRNA and protein expression of ABRACL and CBX4 in human gastric epithelium cell line GES-1 and GC cell lines were assessed with RT-qPCR and western blot. The transfection efficacy of sh-ABRACL, oe-CBX4, and sh-CBX4 was examined with RT-qPCR and western blot. AGS cell proliferation, migration, and invasion were evaluated using CCK-8, colony formation assay, wound healing, and Transwell assays, respectively. With western blot analysis, flow cytometry, and caspase-3 assay kits, the expressions of MMP2 and MMP9, cell apoptosis, and caspase-3 activity were estimated. Western blot was adopted to estimate the contents of apoptosis-related proteins. Luciferase reporter and chromatin immunoprecipitation (ChIP) were applied to verify the interaction between ABRACL and CBX4. The expression of ABRACL and CBX4 was increased in GC tissues and cells. After interfering with ABRACL, the proliferation, migration, and invasion of GC cells were inhibited while apoptosis was promoted. We also discovered that CBX4 could bind to ABRACL and transcriptionally regulate ABRACL expression in AGS cells. Rescue experiments revealed that CBX4 overexpression partially reversed the regulatory effects of ABRACL silencing on the proliferation, migration, invasion, and apoptosis of GC cells. Collectively, ABRACL transcriptionally upregulated by CBX4 promoted the malignant progression of GC.

CMTM4 inhibits gastric tumorigenesis and metastasis.

CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4) is involved in immune regulation and tumor progression; however, its role in gastric cancer (GC) remains unclear. This study explored the role and mechanism of CMTM4 in GC. Immunohistochemistry was used to analyze CMTM4 expression in human gastric biopsied cells from patients with GC (N=23) or chronic superficial gastritis (N=23). To investigate the function of CMTM4 in GC cells, the gene CMTM4 was knocked down and overexpressed in human gastric adenocarcinoma cell line AGS. The gene CMTM4 was overexpressed in AGS cells and human gastric cell line SGC7901. Cell Counting Kit 8 (CCK-8) and cell clonogenic assays were used to analyze the proliferation of the GC cells. Flow cytometry was used to analyze the effects of CMTM4 on apoptosis and the cell cycle. Wound healing and transwell assays were used to analyze the migration and invasion of the gastric cells, respectively. The mechanism of CMTM4 in GC cells was explored using the tandem mass tags (TMTs) proteome and verified by western blot analysis. CMTM4 expression was more downregulated in the human GC tissues than the gastritis tissues. CMTM4 overexpression significantly inhibited the proliferation, migration, and invasion of the GC cells, whereas CMTM4 knockdown enhanced gastric cell proliferation (P>0.05), migration (P>0.05), and invasion (P>0.05). Flow cytometry showed that CMTM4 promoted apoptosis and resulted in G1/S arrest in the GC cells. In addition, the proteome and western blot results showed that STAT1 was significantly upregulated, and the STAT1 signaling pathways were enriched in the GC cells overexpressing CMTM4. Our results suggest that CMTM4 plays a tumor-suppressive role in GC and may affect the growth, migration, and invasion of GC cells through the STAT1 signaling pathway. CMTM4 might have potential value as a prognosis marker and potential therapeutic target for GC therapy.

Activation of the TRPML1 Ion Channel Induces Proton Secretion in the Human Gastric Parietal Cell Line HGT-1.

The lysosomal Ca2+ channel TRPML1 was found to be responsible for gastric acid secretion in murine gastric parietal cells by inducing the trafficking of H+/K+-ATPase containing tubulovesicles to the apical membrane. Therefore, we hypothesized a similar role of TRPML1 in regulating proton secretion in the immortalized human parietal cell line HGT-1. The primary focus was to investigate the involvement of TRPML1 in proton secretion using the known synthetic agonists ML-SA1 and ML-SA5 and the antagonist ML-SI3 and, furthermore, to identify food-derived compounds that target the channel. Proton secretion stimulated by ML-SA1 was reduced by 122.2 ± 22.7% by the antagonist ML-SI3. The steroid hormone 17β-estradiol, present in animal-derived foods, diminished the proton secretory effect of ML-SA1 by 63.4 ± 14.5%. We also demonstrated a reduction in the proton secretory effects of ML-SA1 and ML-SA5 on TRPML1 knock-down cells. The food-derived compounds sulforaphane and trehalose promoted proton secretion in HGT-1 cells but may act independently of TRPML1. Also, histamine- and caffeine-induced proton secretion were affected by neither the TRPML1 antagonist ML-SI3 nor the TRPML1 knock-down. In summary, the results obtained suggest that the activation of TRPML1 promotes proton secretion in HGT-1 cells, but the channel may not participate in canonical signaling pathways.

Syntheses, structures and biological activities of new pyridinyl lactones

Several novel lactones containing either a pyridin-3-yl or a pyridin-2-yl ring in their structure were synthesized: trans δ halolactones 6–8a,b (chloro‑, bromo‑ and iodo-), γ iodolactone 11b and isomeric γ hydroxylactones 9–10a,b. Their structures were confirmed by NMR, IR and HR-MS and additionally by single crystal X-ray diffraction for trans δ bromolactone 6a, trans δ chlorolactone 7b and γ hydroxylactone 10b. Spectral analyses were further supported by DFT calculations. In testing of the compounds for antibacterial activity, none of the lactones exhibited activity against several bacteria strains. However, all lactones exhibited significant cytotoxicity against normal eukaryotic murine fibroblast L929 and human gastric adenocarcinoma AGS cell lines with lactones possessing a pyridine-2-yl ring being more cytotoxic than their pyridine-3-yl counterparts.

Transplanting mitochondria from gastric epithelial cells reduces the malignancy of gastric cancer

Mitochondria are essential for energy supplementation and metabolic homeostasis of cancer cells. Using mitochondria transplantation to reduce the malignancy of gastric cancer (GC) cells is herein proposed. In our study normal human gastric mucous epithelium cell line (GES-1) showed a lower mitochondrial membrane potential (MMP) compared to immortalized human vascular endothelial cell line (EAhy 926) and human gastric adenocarcinoma cell line (AGS). The transplantation of GES-1 mitochondria to AGS were confirmed both by confocal microscopy and flow cytometry. After transplanting GES-1 mitochondria, the AGS showed a reduced cell migration, and invasion without affecting cell viability and apoptosis. Investigating the expression of proteins involved in epithelial-mesenchymal-transition (EMT), transplanted GES-1 mitochondria reduced the expression of mesenchymal markers α-SMA, MMP-9, snail, vimentin and N-cadherin, whereas the epithelial markers E-cadherin and clauding-1 were not changed. The proteins implicated in the cell cycle such as cyclin B1 and D1 were decreased. In mice, inoculation with AGS carrying the transplanted GES-1 mitochondria resulted in smaller sized tumors. Further investigating the mitochondrial balance, the transplanted GES-1 mitochondria were more stably preserved compared to endogenous AGS mitochondria. The MMP, ATP production and mitochondrial mass decreased in GES-1 mitochondria and the mitophagic proteins LC3 II and PINK1 were up-regulated. In conclusion the decreased malignancy of AGS was a result of exogenous GES-1 mitochondria transplantation. This suggests for a therapy with low efficiency mitochondria transplantation in the treatment of cancer cells.

Antiviral Shrimp lncRNA06 Possesses Anti-Tumor Activity by Inducing Apoptosis of Human Gastric Cancer Stem Cells in a Cross-Species Manner.

Virus infection causes the metabolic disorder of host cells, whereas the metabolic disorder of cells is one of the major causes of tumorigenesis, suggesting that antiviral molecules might possess anti-tumor activities by regulating cell metabolism. As the key regulators of gene expression, long non-coding RNAs (lncRNAs) play vital roles in the regulation of cell metabolism. However, the influence of antiviral lncRNAs on tumorigenesis has not been explored. To address this issue, the antiviral and anti-tumor capacities of shrimp lncRNAs were characterized in this study. The results revealed that shrimp lncRNA06, having antiviral activity in shrimp, could suppress the tumorigenesis of human gastric cancer stem cells (GCSCs) via triggering apoptosis of GCSCs in a cross-species manner. Shrimp lncRNA06 could sponge human miR-17-5p to suppress the stemness of GCSCs via the miR-17-5p-p21 axis. At the same time, shrimp lncRNA06 could bind to ATP synthase subunit beta (ATP5F1B) to enhance the stability of the ATP5F1B protein in GCSCs, thus suppressing the tumorigenesis of GCSCs. The in vivo data demonstrated that shrimp lncRNA06 promoted apoptosis and inhibited the stemness of GCSCs through interactions with ATP5F1B and miR-17-5p, leading to the suppression of the tumorigenesis of GCSCs. Therefore, our findings highlighted that antiviral lncRNAs possessed anti-tumor capacities and that antiviral lncRNAs could be the anti-tumor reservoir for the treatment of human cancers.

Metabolic regulator ERRγ governs gastric stem cell differentiation into acid-secreting parietal cells

Parietal cells (PCs) produce gastric acid to kill pathogens and aid digestion. Dysregulated PC census is common in disease, yet how PCs differentiate is unclear. Here, we identify the PC progenitors arising from isthmal stem cells, using mouse models and human gastric cells, and show that they preferentially express cell-metabolism regulator and orphan nuclear receptor Estrogen-related receptor gamma (Esrrg, encoding ERRγ). Esrrg expression facilitated the tracking of stepwise molecular, cellular, and ultrastructural stages of PC differentiation. EsrrgP2ACreERT2 lineage tracing revealed that Esrrg expression commits progenitors to differentiate into mature PCs. scRNA-seq indicated the earliest Esrrg+ PC progenitors preferentially express SMAD4 and SP1 transcriptional targets and the GTPases regulating acid-secretion signal transduction. As progenitors matured, ERRγ-dependent metabolic transcripts predominated. Organoid and mouse studies validated the requirement of ERRγ for PC differentiation. Our work chronicles stem cell differentiation along a single lineage invivo and suggests ERRγ as a therapeutic target for PC-related disorders.

Co-regulatory Proteins Integrate Metabolic Signals to Regulate ESRRG Transcriptional Transactivation in Stomach Parietal Cell Differentiation

Acid-producing stomach parietal cells (PC) are metabolically active and energy demanding cells. Aberrant PC function and census leads to complications ranging from peptic ulcers to stomach adenocarcinoma. Effcient energy expenditure and catabolic metabolism are required for normal PC differentiation from progenitor cells. Here, our data show that the master metabolic transcriptional factor, estrogen-related receptor gamma ( ESRRG), governs murine and human PC differentiation. We are further investigating how known metabolically controlled co-regulatory proteins — Sirtuin 1 (SIRT1), Nuclear Receptor Subfamily 0 Group B Member 2 (NR0B2), and CREB/ATF BZIP Transcription Factor (CREBZF, or SMILE) — integrate stimuli to enable ESRRG transcriptional activation during PC emergence from precursors. Specifically, our lab has developed an acute and reversible PC ablation model to monitor PC recovery in which Simian diphtheria toxin receptor (DTR) expression under the Atp4b promoter targets PC-specific ablation by diphtheria toxin (DT). Additionally, we employ high-dose tamoxifen (HD TAM), which causes a more general gastric injury (independent of tamoxifen effects on estrogen) from PC death along with reactive spasmolytic polypeptide expressing metaplasia (SPEM) of remaining cells. Immunofluorescence, western blotting, and qRT-PCR revealed SIRT1 is upregulated before emergence of early PC clones post-DT and decreases later, suggesting positive regulation of early PC lineage-commitment from stem cells. During the same early time frame, factors NR0B2 and CREBZF decreased, suggesting contrasting roles. Administration of Resv to mice accelerated PC recovery after injury and expanded PCs even at homeostasis. ChIP and ongoing ChIP-seq and Cut&Run assays show ESRRG binds cis-regulatory elements of the metabolic genes and the co-regulatory proteins to promote PC lineage commitment in both mice and in a human gastric cell line in which stable ESRRG-GFP is expressed under a doxycycline-inducible promoter. In other ongoing work, we are generating mice with PC-specific Sirt1/ Nr0b2 ablation to further dissect PC regeneration dynamics and conducting gastric organoid experiments with NR0B2/SIRT1 pharmacological modulators to monitor PC differentiation effects. These organoid samples will be further subjected to epigenetic analysis (Cut&Run, ATACseq) to study chromatin landscape changes. We conclude that cellular metabolic integration by co-regulators enables ESRRG-mediated transactivation during PC differentiation, and SIRT1 promotes early PC lineage-commitment that might offer a promising pro-PC differentiation therapeutic strategy. This work is supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

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.

Abstract 1913: KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with diverse biological functions, including apoptosis, cell survival, inflammation, and immunity. While recombinant human TNFα has demonstrated therapeutic efficacy in isolated limb perfusion for soft tissue sarcoma and melanoma, its systemic administration is limited by severe toxicities, including hypotension. Tumor-specific targeting and enhancement of TNFα's tumor cell killing activity hold promise for improving its therapeutic index. To address these challenges, we developed KD5005, a novel HER-2-targeted immunocytokine fusion protein comprising an anti-HER-2 IgG1 monoclonal antibody (trastuzumab) and human TNFα. Tissue distribution studies revealed that KD5005 specifically accumulated in HER-2-expressing tumors, demonstrating its tumor-targeting capability. In vitro, KD5005 exhibited reduced TNFα-mediated apoptosis in L929 cells compared to recombinant TNFα, indicating its lower non-specific toxicity. Notably, KD5005 induced apoptosis of HER-2-positive human gastric cancer NCI-N87 cells at significantly lower concentrations than trastuzumab, TNFα, or their combination, demonstrating its enhanced tumor cell killing activity. The potent apoptotic activity of KD5005 was HER-2-dependent, as it did not show increased apoptotic activity in WT mouse forestomach carcinoma (MFC) cells and had similar apoptotic activity compared to the combination of trastuzumab and TNFα. This HER-2 dependency further supports the tumor-specific targeting of KD5005.In vivo, KD5005 demonstrated superior anti-tumor efficacy compared to the combination of trastuzumab and TNFα in syngeneic tumor models using CT26 tumor cells transfected with the human HER-2 gene. KD5005 induced robust tumor regression and durable cure in 7 of 8 mice, while the combination of trastuzumab and TNFα elicited weaker therapeutic responses, resulting in tumor rejection in only 3 of 8 mice. Pharmacokinetic and toxicity studies of KD5005 were conducted in cynomolgus monkeys. KD5005 exhibited a significantly prolonged in vivo half-life compared to TNFα, indicating its improved pharmacokinetic profile. KD5005 was administered intravenously every two weeks to the same animal in a dose-escalation study at 0.3 mg/kg, 1.0 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg. The toxicity study results showed that the maximum tolerated dose for KD5005 in cynomolgus monkeys is 10 mg/kg, indicating its favorable toxicity profile. In summary, we have developed KD5005, a HER-2-targeted immunocytokine fusion protein with enhanced tumor-killing activity, improved pharmacokinetic properties, and a favorable toxicity profile. These preclinical findings support the further clinical development of KD5005 as a promising therapeutic agent for HER-2-positive cancers. Citation Format: YI CHEN, PENG LI, GUOBO FANG, HENG WU, CHI ZHANG, ZELING CAI. KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1913.

Abstract 2478: Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity

Abstract Extracellular ATP (eATP) is a potent immunostimulatory signal in the tumor microenvironment (TME), but it is rapidly degraded into AMP and immunosuppressive adenosine. Therefore, ATP metabolism is an attractive anti-tumor target. Several enzymes are involved in the immunosuppressive process used by the TME to convert ATP to AMP and adenosine. Among them, CD39 (gene: ENTPD1), an extracellular ATPase, is a promising target. Analyzing mRNA expression from the TCGA, we show that CD39 is consistently expressed at high levels in comparison to the other extracellular ATP-converting enzymes, ENPP1 and TNAP. We further demonstrate that CD39 has greater enzymatic activity at the slightly acidic physiological pH (pH = 6.8) of the TME than either ENPP1 or TNAP. Flow cytometry profiling of dissociated primary human gastric tumors showed that CD39 was highly expressed on both myeloid and lymphoid cells, including several populations of T cells present in the tumors. This is distinct from findings in healthy primary human PBMCs that show high expression of CD39 on myeloid subsets and relatively few subsets of T cells with high CD39 expression. AB598 is a novel, humanized, Fc-silent anti-CD39 therapeutic antibody that potently binds to CD39 and inhibits its enzymatic activity with sub-nanomolar potency. Treatment of dissociated primary human gastric tumor cells with AB598 resulted in a decrease in the rate of ATP degradation, indicating both the effectiveness of AB598 and the relevance of CD39 as a main contributor to the ATPase activity of the TME. Although eATP is low in healthy tissues, high levels of ATP can be released when cancer cells are treated with immunogenic cell death (ICD)-inducing agents, increasing the eATP in the TME, providing a rationale for the combination of ICD-inducing chemotherapy and AB598. Thus, cancer cells were co-cultured with monocyte-derived dendritic cells (moDCs) and treated with chemotherapy and AB598 to preserve the eATP released by dying cancer cells. The increased expression of CD83 and CD86 on moDCs demonstrated that the combination treatment enhanced the maturation of moDCs, compared to single or no treatment. To further elucidate the anti-tumor effects of the combination treatment, autogenous T cells were introduced into this system. AB598 significantly enhanced T cell activation in the presence of chemotherapy. These data demonstrate that using AB598 results in elevated eATP, which boosts immune cell activation to promote anti-tumor immunity. Citation Format: Ke Jin, Julie Clor, Kaustubh Parashar, Rebecca Fuchs, LC Stetson, Sean Cho, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman. Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2478.

Protective Mechanisms of Various Active Substances on Cell DNA Damage and Apoptosis Induced by Deoxynivalenol.

Deoxynivalenol (DON) is a secondary metabolite of fungi that is harmful to humans and animals. This study examined the protective effects of natural substances, including resveratrol, quercetin, vitamin E, vitamin C, and microbe-derived antioxidants (MA), on both human gastric mucosal cells (GES-1) and pig small intestinal epithelial cells (IPEC-1) when induced by DON. Cells were incubated with active substances for 3 h and then exposed to DON for 24 h. The oxidative stress index, cell cycle, and apoptosis were measured. As compared to cells treated only with DON, pretreatment with active substances improved the balance of the redox status in cells caused by DON. Specifically, quercetin, vitamin E, vitamin C, and MA showed the potential to alleviate the G2 phase cell cycle arrest effect that was induced by DON in both kinds of cells. It was observed that vitamin E and vitamin C can alleviate DON-induced apoptosis and the G2 phase cycle arrest effect mediated via the ATM-Chk 2-Cdc 25C and ATM-P53 signaling pathways in GES-1 cells. In IPEC-1 cells, vitamin C and MA can alleviate both DON-induced apoptosis and the G2 phase cycle arrest effect via the ATM-Chk 2-Cdc 25C signaling pathway. Different bioactive substances utilize different protective mechanisms against DON in interacting with different cells. The proper addition of vitamin E and vitamin C to food can neutralize the toxic effect of DON, while the addition of vitamin C and MA to animal feed can reduce the harm DON does to animals.

Health-Promoting Properties of Processed Red Cabbage (Brassica oleracea var. capitata f. rubra): Effects of Drying Methods on Bio-Compound Retention.

The aim of this work is to describe the effect of convective drying (CD), vacuum drying (VD), infrared drying (IRD), low-temperature vacuum drying (LTVD) and freeze drying (FD) on bio-compound retention of red cabbage and its beneficial health properties. The total phenolics content (TPC), flavonoids (TFC), anthocyanin (TAC) and glucosinolates (TGC) were determined by spectrophotometry. The profiles of phenolic acids, amino acids and fatty acids were determined by HPLC-UV-DAD, LC-DAD and GC-FID, respectively. Antioxidant potential was verified by DPPH and ORAC assays. The antiproliferative activity was measured in the human gastric cell line (AGS). Anti-inflammatory activity was evaluated by phorbol 12-myristate 13-acetate and arachidonic acid models. VD showed high values of TPC = 11.89 ± 0.28 mg GAE/g d.m.; TFC = 11.30 ± 0.9 mg QE/g d.m.; TAC = 0.265 ± 0.01 mg Cya3glu/g d.m.; and TGC = 51.15 ± 3.31 µmol SE/g d.m. Caffeic acid, ferulic acid and sinapic acid were identified. The predominant amino acid and fatty acid were glutamic acid and γ-linolenic acid, respectively. The antioxidant potential was dependent on drying methods for both DPPH and ORAC assays. Dried red cabbage extracts showed clear anti-inflammatory and antiproliferative activity. The dehydration process is an alternative for the retention of bio-compounds and health-promoting properties of red cabbage.

Sodium-Permeable Ion Channels TRPM4 and TRPM5 are Functional in Human Gastric Parietal Cells in Culture and Modulate the Cellular Response to Bitter-Tasting Food Constituents.

Gastric parietal cells secrete chloride ions and protons to form hydrochloric acid. Besides endogenous stimulants, e.g., acetylcholine, bitter-tasting food constituents, e.g., caffeine, induce proton secretion via interaction with bitter taste receptors (TAS2Rs), leading to increased cytosolic Ca2+ and cAMP concentrations. We hypothesized TAS2R activation by bitter tastants to result in proton secretion via cellular Na+ influx mediated by transient receptor potential channels (TRP) M4 and M5 in immortalized human parietal HGT-1 cells. Using the food-derived TAS2R agonists caffeine and l-arginine, we demonstrate both bitter compounds to induce a TRPM4/M5-mediated Na+ influx, with EC50 values of 0.65 and 10.38 mM, respectively, that stimulates cellular proton secretion. Functional involvement of TAS2Rs in the caffeine-evoked effect was demonstrated by means of the TAS2R antagonist homoeriodictyol, and stably CRISPR-Cas9-edited TAS2R43ko cells. Building on previous results, these data further support the suitability of HGT-1 cells as a surrogate cell model for taste cells. In addition, TRPM4/M5 mediated a Na+ influx after stimulating HGT-1 cells with the acetylcholine analogue carbachol, indicating an interaction of the digestion-associated cholinergic pathway with a taste-signaling pathway in parietal cells.

A184 HCPE CONTRIBUTES TO THE HOST INFLAMMATORY RESPONSE TO HELICOBACTER PYLORI

Abstract Background Helicobacter pylori (HP) colonizes chronically 50% of the world population, leading to gastric ulcers or cancers in 2-10 % of infections. Due to rising antibiotic resistance, curtailing HP-induced diseases requires the identification of new anti-HP molecules that can reduce HP viability or its ability to elicit inflammation. Amongst HP’s virulence factors are the Helicobacter Cysteine-rich Proteins (Hcp) that are secreted outside the bacteria and contain Sel-Like Repeats (SLR) which support protein / protein interactions involved in signal transduction across kingdoms. Several SLR-containing bacterial proteins including HcpA are involved in bacteria / host interactions. Thus, Hcps may affect HP’s virulence via interaction of their SLRs with host proteins. Aims We aim to elucidate the role of Hcps in HP’s virulence. Hcps were considered as immune decoys since anti-Hcp antibodies made in infected patients are unable to clear the infection. However, recent data suggest that Hcps interact with host components and may modulate the function of immune cells. However, their effects on gastric cells remain unknown and functional differences between Hcps remain to be explored. Methods This work uses knockout mutagenesis, interactions with gastric cells, transcriptomics, and microscopy. Results We focus on HcpE, the largest Hcp that may interact with many host proteins via its 9 SLRs motifs. We showed that HcpE is secreted outside HP in 2 lab strains and that ~ 30% of secreted HcpE was comprised in outer membrane vesicles that may deliver HcpE into eukaryotic cells by membrane fusion. We also showed that the DiSulfide Bond forming protein DsbK is essential for HcpE production and secretion. Here, we demonstrate the production and secretion of HcpE by clinical isolates in vitro, and its production in situ in infected patients. Using our HcpE and DsbK knockout mutants, we investigate the role of HcpE and other Hcps in interactions with human gastric cells in vitro and in ex vivo gastric explants, showing a role in inflammation activation. Finally, we demonstrate their role in murine gastric colonization and in murine splenocytes activation. Overall, our data show a role of Hcps in host / pathogen interactions largely due to immuno-modulatory functions and a role in gastric colonization, with a predominant role of HcpE over other Hcps. Conclusions The findings open up possibilities to use Hcps or the gate keeper DsbK that controls their production as novel therapeutic targets to alleviate the burden of HP-associated disease.This could benefit the ~6 million patients who develop gastric ulcers or cancers each year worldwide due to HP infection, also saving billions of annual health care costs. Funding Agencies Western University

Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways

ABSTRACT Jaceosidin (JAC) is a natural flavonoid with anti-oxidant and other pharmacological activities; however, its anti-cancer mechanism remains unclear. We investigated the mechanism of action of JAC in gastric cancer cells. Cytotoxicity and apoptosis assays showed that JAC effectively killed multiple gastric cancer cells and induced apoptosis in human gastric adenocarcinoma AGS cells via the mitochondrial pathway. Network pharmacological analysis suggested that its activity was linked to reactive oxygen species (ROS), AKT, and MAPK signaling pathways. Furthermore, JAC accumulated ROS to up-regulate p-JNK, p-p38, and IκB-α protein expressions and down-regulate the p-ERK, p-STAT3, and NF-κB protein expressions. Cell cycle assay results showed that JAC accumulated ROS to up-regulate p21 and p27 protein expressions and down-regulate p-AKT, CDK2, CDK4, CDK6, Cyclin D1, and Cyclin E protein expressions to induce G0/G1 phase arrest. Cell migration assay results showed JAC accumulated ROS to down-regulate Wnt-3a, p-GSK-3β, N-cadherin, and β-catenin protein expressions and up-regulate E-cadherin protein expression to inhibit migration. Furthermore, N-acetyl cysteine pre-treatment prevented the change of these protein expressions. In summary, JAC induced apoptosis and G0/G1 phase arrest and inhibited migration through ROS-mediated signaling pathways in AGS cells.

Single cell atlas of human gastric muscle immune cells and macrophage-driven changes in idiopathic gastroparesis

Gastrointestinal immune cells, particularly muscularis macrophages (MM) interact with the enteric nervous system and influence gastrointestinal motility. Here we determine the human gastric muscle immunome and its changes in patients with idiopathic gastroparesis (IG). Single cell sequencing was performed on 26,000 CD45+ cells obtained from the gastric tissue of 20 subjects. We demonstrate 11 immune cell clusters with Tcells being most abundant followed by myeloid cells. The proportions of cells belonging to the 11 clusters were similar between IG and controls. However, 9/11 clusters showed 578-11,429 differentially expressed genes. In IG, MM had decreased expression of tissue-protective and microglial genes and increased the expression of monocyte trafficking and stromal activating genes. Furthermore, in IG, IL12 mediated JAK-STAT signaling involved in the activation of tissue-resident macrophages and Eph-ephrin signaling involved in monocyte chemotaxis were upregulated. Patients with IG had a greater abundance of monocyte-like cells. These data further link immune dysregulation to the pathophysiology of gastroparesis.

GPR137 inactivates Hippo signaling to promote gastric cancer cell malignancy

As the fifth most common cancer in the world, gastric cancer (GC) ranks as the third major cause of cancer-related death globally. Although surgical resection and chemotherapy still remains the mainstay of potentially curative treatment for GC, chemotherapy resistance and adverse side effects limit their clinical applications. Thus, further investigation of the mechanisms of carcinogenesis in GC and discovery of novel biomarkers is of great concern. We herein report that the elevated expression of GPR137 is correlated with GC. Overexpression of GPR137 potentiates human gastric cancer AGS cell malignancy, including proliferation, migration, invasion, colony formation and xenograft growth in nude mice in vivo, whereas knockout of GPR137 by CRISPR/Cas9 gene editing exerts the opposite effects. Mechanistically, GPR137 could bind to MST, the upstream kinases in Hippo pathway, which disrupts the association of MST with LATS, subsequently activating the transcriptional co-activators, YAP and TAZ, and thereby triggering the target transcription and the alterations in GC cell biological actions consequently. Therefore, our findings may provide with the evidence of developing a potentially novel treatment method with specific target for GC.