MKN45 Gastric Cancer Cells Research Articles

Nuclear factor‑κB inhibitor Bay11‑7082 inhibits gastric cancer cell proliferation by inhibiting Gli1 expression

Dysregulated nuclear factor (NF)-κB signaling pathway is involved in gastric carcinogenesis. The present study aimed to investigate the antitumor effects of the NF-κB inhibitor, Bay11-7082, on gastric cancer (GC) and elucidate its underlying molecular mechanisms. The MTT assay was performed to assess the effects of Bay11-7082 on the proliferation of HGC27 and MKN45 gastric cancer cells. In addition, the Transwell and wound healing assays were performed to determine cell migration and invasion, respectively. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the mRNA and protein expression levels of the target genes. The results demonstrated that the half-maximal inhibitory concentration (IC50) of Bay11-7082 in HGC27 cells was 24.88, 6.72 and 4.23 nM at 24, 48 and 72 h, respectively. Furthermore, the IC50 of Bay11-7082 in MKN45 cells was 29.11, 11.22 and 5.88 nM at 24, 48 and 72 h, respectively. Treatment with Bay11-7082 significantly suppressed the cell migratory and invasive abilities compared with the control group. Notably, Bay11-7082 suppressed GLI Family Zinc Finger 1 (Gli1) mRNA and protein expression levels. Taken together, the results of the present study demonstrated that Bay11-7082 inhibited GC cell proliferation, at least in part through inhibition of Gli1.

Gastrin-17 induces gastric cancer cell epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway.

Gastric cancer (GC) is one of the most common cancers, with most patients often succumbing to death as a result of tumor metastasis. Recent work has demonstrated that gastrin is closely associated with GC metastasis. However, the specific molecular mechanisms underlying this relationship remain to be unveiled. In this study, we assessed the impact of gastrin and the Wnt/β-catenin inhibitor XAV939 on the epithelial-mesenchymal transition (EMT) of the SGC-7901 and MKN45 GC cell lines, and we determined that gastrin-17 significantly decreased E-cadherin expression and upregulated the expression of Snail1 and N-cadherin in GC cells. In addition, gastrin 17 also significantly increased the expression of Wnt3α in a dose-dependent manner. Consistent with these results, gastrin-17 promoted GC cell invasion, proliferation, and migration in a dose-dependent fashion, and these effects were inhibited by XAV939. Together, these results indicated that gastrin-17 induced GC cell EMT, migration, and invasion via the Wnt/β-catenin signaling pathway, which suggests that this gastrin/Wnt/β-catenin signaling axis may represent a therapeutic target for the prevention of GC metastasis.

Laurocerasus officinalis Roem. fruit extract induces cell death through caspase mediated apoptosis in gastric cancer cell lines

Abstract Objectives Laurocerasus officinalis Roem. fruits are traditionally used for several health problems. Although there are some studies about its antiproliferative effects on different cancer cells, no study was reported about its potential therapeutic efficacy against gastric cancers which is the most malignant disease in the digestive system with high morbidity and mortality. Methods This study was aimed to evaluate L. officinalis fruit extract phytochemical contents as well as to compare anticancer effects on gastric cancer cells. The antioxidant activities were determined by ABTS and DPPH assays. Anticancer effects were measured by cell viability assays, then apoptotic proteins were analyzed by western blotting and flow cytometry. Results Laurocerasus officinalis fruit methanol extract showed moderate antioxidant activity by ABTS• and DPPH• assays. Significant cytotoxic activities and caspase mediated apoptosis were detected in the extract treated MKN-45 and AGS gastric cancer cells respectively while sparing healthy cells. Conclusion Our results showed that the L. officinalis Roem. extract has significant anticancer efficacy on gastric cancer cell lines; therefore, it can be further studied to determine its potential therapeutic components.

CGAS regulates the DNA damage response to maintain proliferative signaling in gastric cancer cells.

The activation of some oncogenes promote cancer cell proliferation and growth, facilitate cancer progression and metastasis by induce DNA replication stress, even genome instability. Activation of the cyclic GMP-AMP synthase (cGAS) mediates classical DNA sensing, is involved in genome instability, and is linked to various tumor development or therapy. However, the function of cGAS in gastric cancer remains elusive. In this study, the TCGA database and retrospective immunohistochemical analyses revealed substantially high cGAS expression in gastric cancer tissues and cell lines. By employing cGAS high-expression gastric cancer cell lines, including AGS and MKN45, ectopic silencing of cGAS caused a significant reduction in the proliferation of the cells, tumor growth, and mass in xenograft mice. Mechanistically, database analysis predicted a possible involvement of cGAS in the DNA damage response (DDR), further data through cells revealed protein interactions of the cGAS and MRE11-RAD50-NBN (MRN) complex, which activated cell cycle checkpoints, even increased genome instability in gastric cancer cells, thereby contributing to gastric cancer progression and sensitivity to treatment with DNA damaging agents. Furthermore, the upregulation of cGAS significantly exacerbated the prognosis of gastric cancer patients while improving radiotherapeutic outcomes. Therefore, we concluded that cGAS is involved in gastric cancer progression by fueling genome instability, implying that intervening in the cGAS pathway could be a practicable therapeutic approach for gastric cancer.

Centromere Protein I (CENP-I) Is Upregulated in Gastric Cancer, Predicts Poor Prognosis, and Promotes Tumor Cell Proliferation and Migration.

This study aimed to investigate the expression and cellular function of the centromeric family of proteins (CENPs), especially centromere protein I (CENP-I), in gastric cancer (GC) and identified its clinical significance and cellular functions. CENP-I expression in GC was studied by cDNA microarray, quantitative real-time PCR (qRT-PCR), and immunohistochemistry (IHC), and using datasets from The Cancer Genome Atlas (TCGA), UALCAN, and Gene Expression Omnibus (GEO) databases. Microarray and bioinformatic analyses identified upregulated CENP-A/E/F/H/I/K/P/W and HJURP in stomach adenocarcinoma (STAD), but not in signet ring cell carcinoma (SRCC). Significantly higher CENP-I mRNA expression was also confirmed in 40 pairs of GC tissues than in paired normal gastric tissues by qRT-PCR (P<.001). IHC showed that elevated CENP-I expression was associated with higher tumor stage, lymph node invasion, increased HER2-positive rate (36.7% vs 10.0%), and intestinal Lauren classification in 69 GC samples compared to paired paracancerous normal tissues. The survival of the high-CENP-I group members was poor compared with that of the low-CENP-I group (P = .0011). Cox univariate regression analysis identified tumor size (P = .008), HER2 status (P = .027), and CENP-I expression (P = .049) were independent prognostic factors of GC. The cellular function of CENP-I was studied in MKN45 and MKN28 GC cell lines in vitro. Cell proliferation, migration, and apoptosis were determined using CCK-8, transwell assay, TUNEL assay, and flow cytometry. Our results showed that CENP-I promoted GC cell proliferation, inhibited apoptosis, facilitated cell migration, and induced epithelial–mesenchymal transition (EMT), possibly by activating the AKT pathway. CENP-I expression was correlated with genetic signatures of the proliferative subtype of GC, characterized by intestinal Lauren classification, HER2 amplification, and TP53 mutation. In conclusion, this study revealed an elevated CENP-I expression in GC, which was associated with malignant features and poor prognosis of GC patients, and identified its function in modulating cell proliferation, apoptosis, and migration.

RNF43 and PWWP2B inhibit cancer cell proliferation and are predictive or prognostic biomarker for FDA-approved drugs in patients with advanced gastric cancer

Background: Abnormal regulation of genes has been closely related to gastric cancer. The characterization of gastric cancer has necessitated the development of new therapeutics as well as the identification of prognostic markers to predict the response to novel drugs. In our study, we used RNA sequencing analyses to show that on gastric cancer tissues to identification of gastric cancer prognostic markers. We specifically chose to study RNF43 because it inhibits gastric cancer-related Wnt/β-catenin signaling by interacting with Wnt receptors. PWWP2B was chosen because it is a gene which is downregulated in gastric cancer.Methods: Utilizing RNA sequencing analysis, we evaluated the mRNA expression profile in gastric cancer patients. Also, we used HAP1 cells which is a human near-haploid cell line derived from the male chronic myelogenous leukemia cell line KBM-7. These cell line has one copy of each gene, ensuring the edited allele will not be masked by additional alleles. We investigated the screening of 1,449 FDA-approved drugs in HAP1, HAP1 RNF43 KO and HAP1 PWWP2B KO cells. RNA sequencing data reveals that RNF43 and PWWP2B expression were down-regulated in recurrence gastric cancer patients. Next, we investigated the anti-cancer effects of selected drugs in RNF43 and PWWP2B down-regulated MKN45 gastric cancer cells and xenograft model.Results: Among these FDA-approved drugs, three drugs (docetaxel trihydrate, pelitinib and uprosertib) showed strong inhibitory effects in RNF43 KO cells and PWWP2B KO cells. In MKN45 xenograft model, tumor volumes were significantly reduced in the docetaxel trihydrate, uprosertib or pelitinib-treated group. Our data demonstrated that RNF43 and PWWP2B are a biomarker that predict recurrence of gastric cancer.Conclusions: Our findings suggest that docetaxel trihydrate, uprosertib and pelitinib could be used as novel therapeutic agents for the prevention and treatment of gastric cancer with a decrease in RNF43 and PWWP2B expression.

Disulfiram Chelated With Copper Inhibits the Growth of Gastric Cancer Cells by Modulating Stress Response and Wnt/β-catenin Signaling.

Disulfiram (DSF) is a well-known drug for alcohol abuse. In recent decades, DSF has been demonstrated to exhibit anti-tumor activity; DSF chelated with copper shows enhanced anti-tumor effect. Our goal was to explore the effect of DSF/Cu complex on the growth and metastasis of gastric cancer (GC) in vitro and in vivo. DSF/Cu complex suppressed the proliferation, migration of MKN-45 and BGC-823 GC cells. Furthermore, DSF/Cu treatment reduced the tumor volume in GC mouse models with a tumor suppression rate of 48.24%. Additionally, DSF/Cu induced apoptosis in vitro in MKN-45 and BGC-823 GC cells in a dose- and time-dependent manner as well as in vivo in the xenograft tumor mouse model. Furthermore, DSF/Cu induced autophagy and autophagic flux in MKN-45 and BGC-823 cells, increased the expression of autophagy-related Beclin-1 and LC3 proteins in vivo. Additionally, DSF/Cu suppressed aerobic glycolysis and oxidative phosphorylation by reducing oxygen consumption rate and extracellular acidification rate, respectively, in MKN-45 and BGC-823 cells. Treatment with DSF/Cu induced oxidative stress and DNA damage response by elevating the reactive oxygen species levels; increasing the expression of P53, P21, and γ-H2AX proteins; and inhibiting Wnt/β-catenin signaling in vitro and in vivo. Thus, DSF/Cu suppressed the growth and metastasis of GC cells via modulating the stress response and Wnt/β-catenin signaling. Hence, DSF may be used as a potential therapeutic agent for the treatment of GC.

Overexpression of miRNA-145 induces apoptosis and prevents proliferation and migration of MKN-45 gastric cancer cells.

MiR-145 is a tumor suppressor miRNA that its ubiquitously expressed in the body but in numerous types of cancers such as GC, its expression became reduced or sometimes ceased in many subjects. This study aimed at restoring the function of the miR-145 in MKN-45 cells and investigating the function of this miRNA in proliferation, apoptosis, and migration of GC cells. MKN-45 cells were transfected using the PCMV-miR-145 plasmid vector. The MTT, DAPI staining, and wound healing assays were applied to estimate the impacts of ectopic expression of miR-145 in vitro. Moreover, alterations in the expression levels of K-Ras, c-Myc, caspase-3, caspase-9, Bax, Bcl-2, and MMP-9 mRNA were measured by qRT-PCR analysis. The findings designated that high expression of miR-145 reduced the proliferation and migration and increased the apoptosis of the MKN-45 cells. These effects occur with concurrent suppression of c-Myc, K-Ras, Bcl-2, and MMP-9 as well as induction of caspase-3, caspase-9, and Bax expression. Exogenous miR-145 influences multiple oncogenic pathways and can be regarded as a promising avenue of future therapeutic interventions for GC therapy.

Effects of Toosendanin on the formation of CTPS cytoophidium in human gastric cancer cell MKN-45 and its mechanism

Objective: To investigate the relationship between toosendanin(TSN) and CTP synthase(CTPS) cytoophidium formation in gastric cancer MKN-45 cells. Methods: In this study, the experimental material is MKN-45 human gastric cancer cells. It contains 7 treatment groups of 0, 20, 40, 60, 80, 100, and 120 nmol/L TSN. Each group was treated in triplex privately for 24、48 and 72 hours. Cell counting kit-8 (CCK8) was used to detect the inhibitory effect of TSN on the proliferation of MKN-45 cells. After immunofluorescence detection, the morphology of CTPS cells was observed by a laser confocal microscope. The effect of TSN on MYC gene expression was detected by qRT-PCR. In addition, it contains 2 treatment groups of 1 mmol/L DON and 1 mmol/L MPA, each group was treated in triplex privately for 6 hours and then the cytoophidium morphology was detected by immunofluorescence. Results: The results of immunofluorescence showed that CTPS formed a filamentous cytoophidium structure after treating MKN-45 cells with 1 mmol/L DON and 1 mmol/L MPA, which means that the cells have the ability to form CTPS cytoophidium; The cell proliferation rate of TSN treatment group was significantly lower than that of 0 nmol / L TSN group (P＜0.01); Immunofluorescence results showed that CTPS cytoophidium was the most abundant in MKN-45 cells after treated with 80 nmol/L TSN for 72 h. The results of qRT-PCR showed that MYC expression in cells was significantly decreased after treated with 80 nmol/L TSN for 24 h (P＜0.05), and MYC expression was significantly increased after 48 h (P＜0.01), and then decreased. Conclusion: Toosendanin may affect intracellular cytoophidium assembling by regulating the expression of MYC.

Restoration of miRNA-143 Expression Inhibits Growth and Migration of MKN-45 Gastric Cancer Cell Line.

Purpose: Gastric cancer (GC) is one of the main causes of death from diseases, especially in developing countries. MicroRNAs (miRNAs) are important modulators of the messenger RNAs expression. Among these miRNAs, MiR-143 is a tumor suppressor miRNA and its irregular expression has been revealed in a diversity of malignancies such as GC. Methods: In this study, we have attempted to restore the miR-143 expression in MKN-45 cells by introducing pCMV-miR-143 plasmid vectors. The consequences of exogenous expression of miR-143 on cell proliferation and migration were assessed by MTT and scratch tests, respectively. In addition, the DAPI staining assay was applied for apoptosisquantification. Following miR-143 transfection, the changes in K-Ras, C-Myc, MMP9, Bax, Caspase-3, and Caspase-9 mRNA levels were assessed. Results: The results indicated that the enhanced expression of miR-143 had negative effects on MKN-45 cells proliferation and invasion. Moreover, decreased expressions of K-Ras, MMP9, and C-Myc and up-regulation of Bax, Caspase-3, and Caspase-9 as downstream targets of miR-143 were recognized. Conclusion: These experimental results indicate that reversing the miR-143 expression, by novel techniques, including miRNA replacement could be considered as an efficient approach to reduce cell survival and metastasis.

Curcumin Inhibits Cell Proliferation, Stimulates Apoptosis and Down-Regulates Aldehyde Dehydrogenase Expresion in Gastric cancer Cell line MKN45

According to estimates by the World Health Organization (WHO), in 2018 there were over one million new stomach cancer patients. Vietnam ranks the tenth among the countries with the highest rates of stomach cancer in the world, with the rate of 15.9 cases per 100,000 populations. Research on compounds or drugs that can inhibit cancer cell growth but are less toxic to the body is necessary. In this study, using MTT assays, we have shown that curcumin has ability to inhibit proliferation of stomach cancer cells MKN45. Flow cytometry analysis showed that curcumin increased the percentage of apoptosis cells by 27 - 56% at concentrations of 10 µM - 20 µM and resulted in a typical nuclear morphology of apoptosis. Further, this study showed that curcumin significantly reduced the expression of aldehyde dehydrogenase protein in MKN45 gastric cancer cells. This finding shows that curcumin is a potential therapeutic candidate for gastric cancer cell treatment.

Tamoxifen Downregulates the Expression of Notch1 and DLL1 Genes in MKN-45 Gastric Cancer Cells

Gastric cancer is one of the most prevalent cancers worldwide and the second most common cause for cancer associated mortality. Anti-tumor effects of tamoxifen in breast cancer are well-established. However, no study has so far investigated the effects of tamoxifen on gene expression of Notch1 and DLL1 in gastric cancer cell line. The present study was conducted to explore the effects of tamoxifen, as a repurposed drug, on gene expression of Notch1 and DLL1 in MKN-45, a gastric cancer cell line. MKN-45 cells were cultured in DMEM/F12 medium containing 10% FBS. Cytotoxic effects of tamoxifen on these cells at various concentrations were evaluated by trypan blue exclusion assay. For gene expression analysis, the cells were first incubated with 100 μM tamoxifen followed by total RNA extraction from treated and control cells. Then, cDNA was synthesized. Quantitative real-time PCR using specific primers for Notch1 and DLL1 was performed to assess the effect of tamoxifen on the transcript of them. Treatment with tamoxifen decreased viability of MKN-45 cells in a dose-dependent manner. CC50 was estimated to be around 200 μM. Also, tamoxifen at the dose of 100 μM could significantly downregulate mRNA levels of both Notch1 and DLL1 genes as compared with untreated cells by 24% and 92%, respectively. Based on these results, tamoxifen interferes with Notch signaling pathway through downregulating the expression of Notch1 and DLL1 genes and this could be regarded as a mechanism for its anti-cancer effects in this malignant disease.

Inhibition of NF-κB is required for oleanolic acid to downregulate PD-L1 by promoting DNA demethylation in gastric cancer cells.

Gastric cancer is one of the most common causes of cancer-related death worldwide. Immunotherapy via programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD-L1 expression in gastric cancer cells. Interleukin-1β (IL-1β) at 20 ng/mL was used to stimulate human gastric cancer MKN-45 cells. IL-1β significantly increased PD-L1 expression, which was abolished by OA. Next, OA-treated MKN-45 cells were co-cultured with activated and PD-1-overexpressing Jurkat T cells. OA restored IL-2 levels in the co-culture system and increased T cell killing toward MKN-45 cells. Overexpression of PD-L1 eliminated OA-enhanced T cell killing capacity; however, PD-1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL-1β-increased DNA demethylase activity in MKN-45 cells. DNA methyltransferase inhibitor 5-azacytidine rescued OA-reduced PD-L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD-L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL-1β-treated MKN-45 cells, and overexpression of TET3 restored OA-reduced PD-L1 expression. Finally, OA disrupted nuclear factor κB (NF-κB) signaling IL-1β-treated MKN-45 cells, and overexpression of NF-κB restored OA downregulation of TET3 and PD-L1. The cytotoxicity of T cells toward MKN-45 cells was also weakened by NF-κB overexpression. Altogether, OA blocked the IL-1β/NF-κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD-L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.

Abstract 2524: MiR-29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells as well as on tumor cells

Abstract Background: Peritoneal metastasis (PM) frequently occur in patients with gastric cancer with poor prognosis. Peritoneal cavity is lined with monolayer of mesothelial cells (MC) and contains various types of cells and exosomes. MC play an important role in the development of PM through mesothelial-mesenchymal transition (MMT). MiR-29 family is well known as tumor suppressor. However, the role of miR-29s in PM is not well characterized. We have previously reported that miR-29 family in exosome derived from peritoneal fluid was downregulated in patients with peritoneal metastasis of gastric cancer. In this study, we investigated the functions of miR-29s in the process of peritoneal metastasis. Methods: Exosomes were purified from peritoneal fluids of 85 patients with gastric cancer and miR-29 levels in peritoneal exosomes were quantified with RT-PCR using TaqMan Advanced miRNA Assays. Proliferation and migration of human gastric cancer cell lines, MKN45 and NUGC, were examined with MTT and transwell assays, respectively. Human peritoneal mesothelial cells (HPMCs) were isolated by trypsinization of omental tissues obtained from patients undergoing bariatric surgery and cultured in DMEM supplemented with 20% fetal bovine serum. MiR-29b mimic and negative control were transfected by lipofection method using RNAiMAX. MMT was induced by the culture of HPMCs with 10 ng/ml TGF-β. Expressions of cytokeratin and vimentin were evaluated with flow cytometry. Results: The expressions of miR-29a, miR-29b and miR-29c in peritoneal exosomes were all downregulated in 33 patients with PM as compared to those without PM. Transfection of miR-29b mimic significantly decreased the proliferation of MKN45 and NUGC-4 by 22~58% as compared with negative control miRNA (p&amp;lt;0.01). Migration was inhibited more strongly by 84~90% (p&amp;lt;0.01). After 48 hour-culture with TGF-β, HPMCs apparently changed the morphology from round to spindle shape. The expression level of cytokeratin in HPMCs was decreased while vimentin tended to be upregulated by TGF-β. However, transfection of miR-29b, but not control miRNA, significantly suppressed the morphological changes and reduced the expression levels of vimentin with restored expression of cytokeratin, suggesting the inhibition of MMT in HPMCs. Conclusion: MiR-29b suppresses mesenchymal transition of MC as well as of tumor cells. Replacement of miR-29b to peritoneal cavity might be used for the treatment for peritoneal metastasis. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Akira Saito, Naohiro Sata, Joji Kitayama. MiR-29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells as well as on tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2524.

Lentinan, a Shiitake Mushroom ß-Glucan, Downregulates the Enhanced PD-L1 Expression Induced by Platinum Compounds in Gastric Cancer Cells -

Background: Despite recent therapeutic improvements, the prognosis of unresectable gastric cancer remains poor. Upregulation of programmed cell death ligand 1 (PD-L1) in tumor cells is believed to be an important mechanism to escape from the host immune response. The expression of PD-L1 in tumors is regulated in a highly complex manner by various upstream signaling molecules, depending on the cell type. Given that the efficacy of chemotherapeutic agents for metastatic gastric cancer is limited due to immune escape caused by enhanced PD-L1 expression, PD-1/PD-L1 targeted immunotherapy may be a promising alterative for chemotherapy. However, immune checkpoint inhibitor monotherapy has shown clinical benefits in less than 20% of patients with gastric cancer and its underlying mechanism remains to be elucidated. On the other hand, lentinan, a glucan purified from Shiitake mushrooms, has significant immune-stimulating effects and has been reported to improve survival in patients with metastatic gastric cancer receiving chemotherapy. In the current study we investigated the mechanism by which lentinan increases the chemotherapeutic efficacy by focusing on the expression of PD-L1. Methods: To evaluate the effects of lentinan as well as antineoplastic agents, the expression of PD-L1 and associated molecules was analyzed by real-time polymerase chain reaction and western blotting using the human gastric cancer cell lines, NUGC3, MKN1, and MKN45. Results: Treatment with either cisplatin or oxaliplatin dose-dependently enhanced PD-L1 mRNA and protein expression through the mitogen-activated protein kinase (MAPK) pathway in gastric cancer cells. However, lentinan treatment inhibited the platinum drug-stimulated expression of PD-L1 in gastric cancer cells mainly by suppressing MAPK signaling without affecting the phosphatidylinositol-3 kinase/AKT pathway or transcription factors. Conclusions: Platinum-based drugs enhanced the expression of PD-L1 via the MAPK pathway in gastric cancer cells. Lentinan downregulated PD-L1 expression induced by either cisplatin or oxaliplatin, suggesting that a combination of this glucan and platinum-based chemotherapy could restore the chemosensitivity of cells.

The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker β-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.

PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

PIK3CA mutations are frequently observed in various human cancers including gastric cancer (GC). This study was conducted to investigate the anti-tumor effects of alpelisib, a PI3K p110α-specific inhibitor, using preclinical models of GC. In addition, the combined effects of alpelisib and paclitaxel on GC were evaluated. Among the SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1 GC cells, three PIK3CA-mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 phase arrest regardless of PIK3CA mutational status. The alpelisib and paclitaxel combination demonstrated synergistic anti-proliferative effects, preferentially on PIK3CA-mutant cells, resulting in increased DNA damage response and apoptosis. In addition, alpelisib and paclitaxel combination potentiated anti-migratory activity in PIK3CA-mutant cells. Alpelisib partially reversed epithelial–mesenchymal transition markers in PIK3CA-mutant cells. In a xenograft model of MKN1 cells, the alpelisib and paclitaxel combination significantly enhanced anti-tumor activity by decreasing Ki-67 expression and increasing apoptosis. Moreover, this combination tended to prolong the survival of tumor-bearing mice. Our data suggest promising anti-tumor efficacy of alpelisib alone or in combination with paclitaxel in PIK3CA-mutant GC cells.

Distinct prognostic values and antitumor effects of tumor growth factor β1 and its receptors in gastric cancer.

Gastric cancer (GC) is one of the most common malignancies and is the second leading cause of cancer-associated mortality world-wide. In the present study, the prognostic value and antitumor effects of transforming growth factor β1 (TGFβ1) and its receptors in GC were explored. The online Kaplan-Meier plotter database was used to investigate the prognostic values of TGFβ1 and its receptors. The present study demonstrated that low mRNA expression levels of TGFβ1 and its 3 receptors, transforming growth factor β1 (TGFβR1), TGFβR2 and TGFβR3, was associated with improved overall survival time in patients with GC. Cell Counting Kit-8 and Transwell assays were used to confirm the effects of TGFβ1, TGFβR1, TGFβR2 and TGFβR3 on the proliferation, migration and invasiveness of the AGS and MKN45 GC cell lines. It was found that the knockdown of these genes blocked cell proliferation, migration and invasion in GC cells. To the best of our knowledge, the present study is the first to determine the role of TGFβR1 and TGFβR3 in GC cells. The results indicate that in addition to TGFβ1 and TGFβR2, TGFβR1 also plays a specific role in the occurrence and development of tumors. Thus, these markers may be considered as potential prognostic indicators in human GC. The findings of the present study indicate that not only TGFβ1 and TGFβR2, but also TGFβR1 is involved in the progression of GC. The findings of the present study provide new ideas and approaches for the treatment of patients with GC.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

FAM83H and SCRIB stabilize β-catenin and stimulate progression of gastric carcinoma.

FAM83H primarily is known for its function in tooth development. Recently, a role for FAM83H in tumorigenesis, conjunction with MYC and β-catenin, has been suggested. Analysis of public data indicates that FAM83H expression is closely associated with SCRIB expression in human gastric cancers. Therefore, this study investigated the roles of FAM83H and SCRIB in 200 human gastric cancers and gastric cancer cells. In human gastric carcinomas, both the individual and combined expression patterns of the nuclear FAM83H and SCRIB were independent indicators of shorter survival of gastric carcinoma patients. In MKN-45 and NCI-N87 gastric cancer cells, the expression of FAM83H and SCRIB were associated with proliferation and invasiveness of cells. FAM83H-mediated in vivo tumor growth was attenuated with knock-down of SCRIB. Moreover, immunoprecipitation indicates that FAM83H, SCRIB, and β-catenin, form a complex, and knock-down of either FAM83H or SCRIB accelerated proteasomal degradation of β-catenin. In conclusion, this study has found that the individual and combined expression patterns of nuclear FAM83H and SCRIB are prognostic indicators of gastric carcinomas and further suggests that FAM83H and SCRIB are involved in the progression of gastric carcinomas by stabilizing β-catenin.