MKN45 Gastric Cancer Cells Research Articles

Mesenchymal stem cell-derived CXCL16 promotes progression of gastric cancer cells by STAT3-mediated expression of Ror1.

Bone marrow‐derived mesenchymal stem or stromal cells (MSC) have been shown to be recruited to various types of tumor tissues, where they interact with tumor cells to promote their proliferation, survival, invasion and metastasis, depending on the type of the tumor. We have previously shown that Ror2 receptor tyrosine kinase and its ligand, Wnt5a, are expressed in MSC, and Wnt5a‐Ror2 signaling in MSC induces expression of CXCL16, which, in turn, promotes proliferation of co–cultured MKN45 gastric cancer cells via the CXCL16‐CXCR6 axis. However, it remains unclear how CXCL16 regulates proliferation of MKN45 cells. Here, we show that knockdown of CXCL16 in MSC by siRNA suppresses not only proliferation but also migration of co–cultured MKN45 cells. We also show that MSC‐derived CXCL16 or recombinant CXCL16 upregulates expression of Ror1 through activation of STAT3 in MKN45 cells, leading to promotion of proliferation and migration of MKN45 cells in vitro. Furthermore, co–injection of MSC with MKN45 cells in nude mice promoted tumor formation in a manner dependent on expression of Ror1 in MKN45 cells, and anti–CXCL16 neutralizing antibody suppressed tumor formation of MKN45 cells co–injected with MSC. These results suggest that CXCL16 produced through Ror2‐mediated signaling in MSC within the tumor microenvironment acts on MKN45 cells in a paracrine manner to activate the CXCR6‐STAT3 pathway, which, in turn, induces expression of Ror1 in MKN45 cells, thereby promoting tumor progression.

Involvement of ribosomal protein L11 expression in sensitivity of gastric cancer against 5-FU.

5-Fluorouracil (5-FU) is widely used in the treatment of various types of solid cancer. Our study showed that ribosomal protein L11 (RPL11) was a crucial factor affecting sensitivity of gastric cancer to 5-FU, implying that RPL11 expression is a potential biomarker for predicting 5-FU sensitivity. Kaplan-Meier survival analysis indicated that high RPL11 expression in gastric cancer patients treated with 5-FU was significantly associated with good prognosis. It was therefore investigated whether RPL11 affected the sensitivity of gastric cancer against 5-FU using four human gastric cancer cell lines, MKN45 (wild-type TP53 gene), NUGC4 (wild-type), MKN7 (mutated), and KE39 cells (mutated). In vitro assays demonstrated that RPL11 knockdown in gastric cancer cell lines carrying the TP53 wild-type gene attenuated 5-FU-induced cell growth suppression and activation of the P53 pathway, but not in cells carrying mutated TP53, suggesting that 5-FU suppresses tumor progression via RPL11-mediated activation of the P53 pathway in gastric cancer. The present study provides a potential therapeutic strategy for improving 5-FU resistance in gastric cancer by elevating RPL11 expression.

Analysis of the Effect of Increased α2,3-Sialylation on RTK Activation in MKN45 Gastric Cancer Spheroids Treated with Crizotinib.

In the scenario of personalized medicine, targeted therapies are currently the focus of cancer drug development. These drugs can block the growth and spread of tumor cells by interfering with key molecules involved in malignancy, such as receptor tyrosine kinases (RTKs). MET and Recepteur d’Origine Nantais (RON), which are RTKs frequently overactivated in gastric cancer, are glycoprotein receptors whose activation have been shown to be modulated by the cellular glycosylation. In this work, we address the role of sialylation in gastric cancer therapy using an innovative 3D high-throughput cell culture methodology that mimics better the in vivo tumor features. We evaluate the response to targeted treatment of glycoengineered gastric cancer cell models overexpressing the sialyltransferases ST3GAL4 or ST3GAL6 by subjecting 3D spheroids to the tyrosine kinase inhibitor crizotinib. We show here that 3D spheroids of ST3GAL4 or ST3GAL6 overexpressing MKN45 gastric cancer cells are less affected by the inhibitor. In addition, we disclose a potential compensatory pathway via activation of the Insulin Receptor upon crizotinib treatment. Our results suggest that cell sialylation, in addition of being involved in tumor progression, could play a critical role in the response to tyrosine kinase inhibitors in gastric cancer.

Lemon Peel Polyphenol Extract Reduces Interleukin-6-Induced Cell Migration, Invasiveness, and Matrix Metalloproteinase-9/2 Expression in Human Gastric Adenocarcinoma MKN-28 and AGS Cell Lines.

Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon (Citrus limon) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5–1 μg/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer.

Calreticulin regulates vascular endothelial growth factor-A mRNA stability in gastric cancer cells.

Calreticulin (CRT) and vascular endothelial growth factor-A (VEGF-A) are crucial for angiogenesis, and mediate multiple malignant behaviors in gastric cancer. In this study, we report that CRT is positively correlated with VEGF-A in gastric cancer patients. Moreover, high expressions of both CRT and VEGF-A are markedly associated with the pathological stage, progression, and poor prognosis in the patients. Therefore, we sought to elucidate the mechanism by which CRT affects VEGF-A in gastric cancer. Firstly, we demonstrate the novel finding that knockdown of CRT reduced VEGF-A mRNA stability in two gastric cancer cell lines, AGS and MKN45. The AU-Rich element (ARE) is believed to play a crucial role in the maintenance of VEGF-A mRNA stability. Luciferase reporter assay shows that knockdown of CRT significantly decreased the activity of renilla luciferase with VEGF-A ARE sequence. Additionally, competition results from RNA-binding/electrophoretic mobility shift assay indicate that CRT forms an RNA-protein complex with the VEGF-A mRNA by binding to the ARE. In addition, the proliferation rate of human umbilical vein endothelial cells (HUVEC) was significantly reduced when treated with conditioned medium from CRT knockdown cells; this was rescued by exogenous VEGF-A recombinant protein. Our results demonstrate that CRT is involved in VEGF-A ARE binding protein complexes to stabilize VEGF-A mRNA, thereby promoting the angiogenesis, and progression of gastric cancer.

7-H-Pyrrolo[2,3-d]pyrimidine derivative acts as promising agent for gastric cancer treatment by inducing cell death.

In the present study effect of 7-H-pyrrolo[2,3-d]pyrimidine derivative (7-HPPD) on viability of MKN28 and MKN74 gastric cancer cells was investigated. There was no significant change in GES-1 cell viability on treatment with 7-HPPD for 48h. MKN28 and MKN74 cell viability was reduced to 21 and 23%, respectively, on treatment with 7-HPPD at concentration of 50µM. Hoechst 33342 staining showed that the cells treated with 7-HPPD showed condensation of chromatin material, presence of apoptotic bodies and intense blue fluorescence. Treatment of MKN28 and MKN74 cells with 7-HPPD markedly increased the release of LDH. Z-VAD-FMK prevented 7-HPPD-induced suppression of MKN28 and MKN74 cell viability. Exposure to 15, 20, 25, 30 and 50µM concentrations of 7-HPPD caused concentration-based increase in caspase-8, -9, -3 and cleaved PARP. A significant increase in ROS production was caused by 7-HPPD in MKN28 and MKN74 cells. Increasing the concentration of 7-HPPD from 10 to 50µM did not increase the expression of RIP3 protein. In summary, 7-HPPD suppresses gastric cancer cell growth by inducing apoptosis through increase in caspase expression and ROS production. Consequently, 7-HPPD may be used for the development of treatment strategy for gastric cancer.

Calcium-sensing receptor bridges calcium and telomerase reverse transcriptase in gastric cancers via Akt.

Human telomerase reverse transcriptase (hTERT) and calcium-sensing receptor (CaSR) act as an oncogene in gastric cancers, however, their relationship in the progression of gastric cancers is yet to be elucidated. Herein, we aimed to access the potential interaction between hTERT and CaSR in the development of gastric cancers. The clinical data of 41 patients with gastric cancers were analyzed regarding the expressions of hTERT and CaSR by immunohistochemistry. Among them, five patients' specimens were also analyzed by Western blotting. The regulation of calcium on the expression level of hTERT and the possible underlying mechanism via CaSR were explored in gastric cancer cell lines MKN45 and SGC-7901. Both hTERT and CaSR were increased and positively correlated in human gastric cancers, which also occurs in gastric cancer cells MKN45 and SGC-7901. Calcium induced hTERT expression at the transcriptional level in a CaSR-dependent manner followed by an increase in telomerase activity, as either a CaSR shRNA or the CaSR antagonist NPS2143 abolished the calcium-mediated regulation of hTERT and telomerase activity. Further studies showed that CaSR-mediated cytosolic calcium rise followed by Akt activation was involved in the regulation of hTERT by extracellular calcium. Finally, neither CaSR overexpression nor shRNA-mediated CaSR downregulation had an effect on the expression level of hTERT. Our findings established a functional linkage between CaSR and hTERT in the development of gastric cancers and CaSR-hTERT coupling might serve as a novel target for therapeutic strategy against human gastric cancers.

Schisantherin A induces cell apoptosis through ROS/JNK signaling pathway in human gastric cancer cells

Gastric cancer is one of the most lethal cancers with unmet clinical treatment and low 5-year survival rate. Schisantherin A is a major compound derived from Fructusschisandrae while its anti-tumor role remains nearly unknown. Here, we reported that schisantherin A had an anti-proliferation effect on gastric cancer cell lines MKN45 and SGC-7901. Schisantherin A induced cell cycle arrest at G2/M phase and cell apoptosis, and inhibited cell migration in gastric cancer MKN45 and SGC7901 cells. Meanwhile, upregulation of cleaved caspase-9, cleaved caspase-3 and cleaved PARP were accompanied with the loss of mitochondrial membrane potential (MMP). Moreover, schisantherin A induced ROS-dependent JNK phosphorylation with higher ROS production. The JNK inhibitor and ROS scavenger NAC rescued the cell apoptosis and cycle inhibition elicited by schisantherin A. Furthermore, the expression level of antioxidant factor Nrf2 was suppressed by schisantherin A. These findings suggest that schisantherin A possesses an anti-tumor activity via activation of ROS/JNK with Nrf2 inhibition, indicating that schisantherin A is a promising chemotherapeutic candidate for gastric cancer.

Propofol suppresses proliferation, migration and invasion of gastric cancer cells via regulating miR-29/MMP-2 axis.

Propofol (2,6-diisopropylphenol) is a commonly used intravenous anesthetic agent. Previous studies suggested that propofol might act as anti-tumor drug in various cancers, including gastric cancer. However, the underlying mechanism is still largely unknown. 1, 5, 10 and 20 μg/ml of propofol were used to treat gastric cancer cell MKN45 for 24, 48 or 72 hours. MTT assay was used to detect the proliferation. Transwell assay was employed to measure the invasion and migration with or without matrigel. The expression of miR-29a, 29b and 29c was assessed by quantitative real time polymerase chain reaction (qRT-PCR). Luciferase assay was introduced to confirm the relationship between miR-29 family member and MMP-2. Western blot was adopted to measure the expression of MMP-2 protein. The proliferation, migration and invasion of gastric cancer cell MKN45 were gradually decreased after propofol treatment in time- and dose- dependent manners. MiR-29a, b and c were downregulated in MKN45 cells compared with normal gastric mucosa epithelial cell GES-1 and upregulated by propofol. Inhibition of miR-29a, b or c promoted cell proliferation, migration and invasion of MKN45 cells under propofol treatment. MMP-2 was a target and regulated by miR-29 family and propofol. MMP-2 silencing reversed the stimulative effects of miR-29 inhibitor. Propofol inhibited cell proliferation, migration and invasion by upregulating miR-29a, miR-29b and miR-29c and downregulating MMP-2.

MiR-4766-5p Inhibits The Development And Progression Of Gastric Cancer By Targeting NKAP.

PurposeIt is widely known that some specific microRNAs can regulate the expressions of genes in gastric cancer cells at the post-transcriptional level. Previous studies have identified that miRNA-4766-5p was involved in tumor cell proliferation and can be an independent prognostic indicator for malignant pleural mesothelioma. However, the mechanism underlying gastric cancer via the miRNA-4766-5p pathway remains to be blank.MethodsWe investigated the expression of miR-4766-5p in gastric cancer tissues and cells through qRT-PCR. We used RNAi to change the expressions of miR-4766-5p in gastric cancer cell lines, AGS and MKN45. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the mRNA expression of miR-4766-5p. We identified cell proliferation by CCK8 and clone formation assays. We analyzed the cell apoptosis and cycle through flow cytometry. At last, we used a dual-luciferase reporter assay to illustrate the interaction between miR-4766-5p and NKAP and used Western blot to determine the protein expression of signaling pathways.ResultsWe found that 1) miR-4766-5p was down-regulated in gastric cancer tissues and cells lines; 2) miR-4766-5p inhibited cell proliferation of gastric cancer cell lines significantly; 3) miR-4766-5p significantly inhibited cell migration and invasion of gastric cancer cells; 4) miR-4766-5p induced gastric cancer cell apoptosis. 5) NKAP was a direct target gene of miR-4766-5p; and 6) miR-4766-5p induced inactivation of AKT/mTOR pathway.ConclusionThe above results indicate that miR-4766-5p suppressed the proliferation and metastasis of gastric cancer cells through targeting NKAP. Our findings could probably contribute to the diagnostics and prognostics of gastric cancer through new methodologies.

HER2 Regulates Cancer Stem Cell Activities via the Wnt Signaling Pathway in Gastric Cancer Cells

Introduction: Human epidermal growth factor 2 (HER2) gene overexpression in breast carcinoma cell lines has been shown to drive mammary carcinogenesis and tumor growth and invasion through its effects on mammary stem cells. Objective: Therefore, we investigated the mechanism by which HER2 regulates cancer stem cell (CSC) activity in gastric cancer cells. Methods: HER2 was transfected into MKN28 gastric cancer cells, and its role in regulating CSC activity was determined by characterizing the HER2-overexpressing cells. Results: The sphere formation assay revealed that the sphere sizes and frequency of sphere formation were significantly greater for the HER2-overexpressing cells than for the MKN28 control cells. The CSC markers Oct-4 and BMI1 were more highly expressed in the HER2-overexpressing cells, as were the EMT markers. This was accompanied by a significant enhancement in cellular invasion of the Matrigel and migration. The E-cadherin level was significantly downregulated, and the mesenchymal marker Snail upregulated, in the HER2-transfected cells. HER2 overexpression activated the well-characterized CSC-associated Wnt/β-catenin signaling pathway, as shown by the luciferase assay. After treatment of these cells with the Wnt signal inhibitor PRI-724, the BMI1 and Oct-4 levels were decreased for 24 h and Snail was also downregulated. Immunofluorescence staining revealed the significant restoration of E-cadherin levels in the HER2-transfected cells after PRI-724 treatment. Conclusions: These results established a role for HER2 in regulating gastric CSC activity, with Wnt/β-catenin signaling being mediated via a HER2-dependent pathway. In summary, HER2-overexpressing gastric cancer cells exhibited increased stemness and invasiveness and were regulated by Wnt/β-catenin signaling.

Three Cd(II) coordination polymers induce gastric cancer cell apoptosis by promoting intracellular ROS accumulation and apoptotic genes expression

In this study, three new Cd(II) coordination polymers, [Cd(TPT)(NO3)2(MeOH)](CH2Cl2) (1), [Cd3(TPT)2(NO3)6(MeOH)4](MeOH)2 (2) and [Cd(TPT)(NO3)2] (3), with structural diversity [from one-dimensional (1D) chain to 3D framework structures] have been prepared by using 2,4,6-tris-(pyridyl)-1,3,5-triazine (TPT) as ligand. The CCK-8 assay was used to explore the anti-proliferation effect of 1–3 on MKN45 gastric cancer cells, Annexin V-FITC/PI assay was performed to confirm the apoptosis and cell cycle arrest of MKN45 in a flow cytometer; the intracellular ROS level was also determined in this study. mRNA expressions of p53, nf-κb, and tnf-α in MKN45 cancer cells after treatment with 1–3 was measured by quantitative reverse transcription polymerase chain reaction. The biological results showed that 1 exerted anticancer activity through inducing apoptosis by upregulating the apoptotic genes and intracellular ROS level.

Development of a novel class of peroxisome proliferator-activated receptor (PPAR) gamma ligands as an anticancer agent with a unique binding mode based on a non-thiazolidinedione scaffold

We previously identified dibenzooxepine derivative 1 as a potent PPARγ ligand with a unique binding mode owing to its non-thiazolidinedione scaffold. However, while 1 showed remarkably potent MKN-45 gastric cancer cell aggregation activity, an indicator of cancer differentiation-inducing activity induced by PPARγ activation, we recognized that 1 was metabolically unstable. In the present study, we identified a metabolically soft spot, and successfully discovered 3-fluoro dibenzooxepine derivative 9 with better metabolic stability. Further optimization provided imidazo[1,2-a]pyridine derivative 17, which showed potent MKN-45 gastric cancer cell aggregation activity and excellent PK profiles compared with 9. Compound 17 exerted a growth inhibitory effect on AsPC-1/AG1 pancreatic tumor in mice. Furthermore, the decrease in the hematocrit (an indicator of localized edema, a serious adverse effect of PPARγ ligands) was tolerable even with oral administration at 200 mg/kg in healthy mice.

Effects of dihydromyricetin on the migration and invasion of human gastric cancer MKN45 cells and its mechanism

To investigate the effects of dihydromyricetin (DHM) on the migration and invasion of human gastric cancer MKN45 cells and its mechanism and provide experimental basis for the prevention and treatment of gastric cancer with Traditional Chinese Medicine (TCM). MKN45 cells were pre-treated with DHM (0,10,20,30,40,50 μmol/L) for 24 and 48 hours respectively. Cell viability treated with different concentrations of DHM was detected by Cell Counting kit (CCK-8) assay, cell migration was measured by wound healing assay, and cell invasion was tested by Transwell assay. Cells were pre-treated with DHM or co-treated with c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, then, the levels of migration- and invasion-related proteins were tested by Western blot. DHM concentration-dependently inhibited cell migration and invasion and downregulated matrix metalloprotein -2 (MMP-2) and phosphorylated JNK (pJNK) expression in MKN45 cells, followed by upregulation of E-cadherin and downregulation of Vimentin. Co-treatment with DHM and JNK inhibitor SP600125 further suppressed MMP-2 expression and cell invasion in MKN45 cells, suggesting that DHM inhibited MKN45 cells metastasis through JNK/MMP-2 pathway. DHM can inhibit cell migration and invasion in human gastric cancer MKN45 cells through downregulating MMP-2 expression via JNK signaling pathway and reverse epithelial-mesenchymal transition (EMT), implying that DHM could have the potential to serve as an anti-metastatic agent for treating gastric cancer.

Over expression of amphiregulin promoted malignant progression in gastric cancer

Human Amphiregulin (AREG) was found to be over expressed in many types of cancer patients including gastric cancer (GC). However, its role in GC development remained unknown. In this study, over expression of AREG in MGC803 and SGC7901 GC cells were conducted by gene transfection through utilizing gateway recombinant cloning technology. Meanwhile, AREG knocked down in BGC823 and MKN45 GC cells were achieved by lentvirus-mediated shRNA oligos infection. The results showed that over expression of AREG promoted cancer cells proliferation, invasion and migration, prevented apoptotic cell death and facilitated cell cycle progression. However, the above AREG-mediated oncogenic phenotypes were reversed while AREG was knocked down in cancer cells. Furthermore, the expression of AREG in GC cells was associated with higher level of activation of both ERK/JNK/p38 and PI3K/Akt signaling pathways, which were also diminished by AREG knocked down. Finally, the result from study in vivo showed that the silencing AREG expression by AREG knocked down inhibited tumor growth in nude mice. Collectively, the data provided a convincingly laboratory evidence that over expression of AREG promoted malignant procession via activation of ERK/JNK/p38 and PI3K/Akt signaling pathways. Thus AREG possessed an oncogenic activity and could be served as a target for gastric cancer therapy.

DHA Abolishes the Detrimental Effect of Docetaxel on Downregulation of the MICA via Decreasing the Expression Level of MicroRNA-20a in Gastric Cancer.

MHC class I chain-related protein A (MICA) is a membrane glycoprotein expressed abnormally on some malignant cells including gastric cancer (GC) cell and elicits anti-tumor immune responses. Downregulation of MICA expression could lead to immune-evasion of cancer cells. In this study, we aimed to investigate the effect of docosahexaenoic acid (DHA) and docetaxel alone or in combination on the expression level of MICA and its regulating microRNA (miRNA), miR-20a in MKN45 GC cell line. MKN45 GC cell line was cultured and MTT assay was performed to determine IC50 of docetaxel. Cells were treated by 18.5μM docetaxel and 100μM DHA. After that, RNA extraction and cDNA synthesis were done and the expression level of MICA and miR-20a were determined by quantitative real-time PCR for both treated and untreated cell lines. Our findings showed less downregulation of the expression level of MICA by the combination of docetaxel/DHA (5.34-fold) compared with docetaxel (45.45-fold) and DHA (55.55-fold). Consistently, combination therapy led to the more downregulation of the expression level of the miR-20a (5.20-fold) in comparison to docetaxel (2.38-fold) and DHA (1.60-fold). As an unwanted effect of docetaxel therapy in GC, downregulation of MICA expression could lead to weak anti-tumor immune responses. By increasing the expression level of MICA, combination therapy of docetaxel with DHA would be useful to overcome this side effect.

Genipin induces mitochondrial dysfunction and apoptosis via downregulation of Stat3/mcl-1 pathway in gastric cancer

BackgroundGenipin is a compound derived from gardenia fruit extract. Although Genipin has anti-tumor effects in various cancers, its effect and mechanism in gastric cancer remain unclear. Here, we investigated the relationship between the anticancer effect of Genipin and signal transducer and activator of transcription (Stat3)/myeloid cell leukemia-1 (Mcl-1) in human gastric cancers.MethodsMTT assays were performed to determine the cell viability of gastric cancer and gastric epithelial cell lines (AGS, MKN45, SNU638, MKN74, HFE-145). A TUNEL assay and Western blotting were carried out to investigate apoptosis. Stat3 activity was measured by proteome profiler phospho kinase array, immunofluorescence and immunoblotting. Mitochondria function was monitored with an XF24 analyzer and by flow cytometry, confocal microscopy using fluorescent probes for general mitochondrial membrane potential (MMP).ResultsGenipin induced apoptosis in gastric cancer cells, including AGS and MKN45 cells. Genipin also reduced Mcl-1 mRNA and protein levels. Furthermore, we found that phosphorylation of Stat3 is regulated by Genipin. Additionally, the protein level of phospho Janus kinase 2 (JAK2) was decreased by Genipin treatment, indicating that the Stat3/JAK2/Mcl-1 pathway is suppressed by Genipin treatment in gastric cancer cells. Mcl-1 is closely related to mitochondrial function. These findings suggest that Genipin contributes to the collapse of mitochondrial functions like MMP.ConclusionsGenipin induced apoptosis by suppressing the Stat3/Mcl-1 pathway and led to mitochondrial dysfunction. Our results reveal a novel mechanism for the anti-cancer effect of Genipin in gastric cancer.

Altered Expression of CD44, SIRT1, CXCR4, miR-21, miR-34a, and miR-451 Genes in MKN-45 Cell Line After Docetaxel Treatment.

Today it is known that the gene expression profile of cancer stem cells differs from other cancer cells, which may lead to the resistance to routine treatments. The aim of this study was to investigate the effect of docetaxel (DOC) treatment on CD44+ cell frequency in human gastric cancer (GC) MKN-45 cell line and its effect on expression levels of SIRT1, CXCR4, microRNA (miR)-21, miR-451, and miR-34a that are closely correlated with the chemoresistance or self-renewal of cancer stem cells (CSCs). The cytotoxic effect of DOC on MKN-45 cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-assay. The frequency of CD44+ cells was measured by flow cytometry in the treated and control groups. The expression level of SIRT1, CXCR4, miR-21, miR-451, and miR-34a was assessed in DOC-treated and non-treated cells using quantitative real-time PCR. Data were analyzed using Statistical Package for the Social Sciences (SPSS) software. The half-maximal inhibitory concentration (IC50) of DOC was 10μg/ml after 48h. Flow cytometry showed a significant increase in CD44+ cells after treatment with DOC (94.3%) when compared with non-treated cells (84.6%) (P < 0.01). The expression of SIRT1, CXCR4, and miR-21 was up-regulated (1.4-fold, 6.7-fold, and 1.22-fold, respectively, P < 0.05) in DOC-treated cells relative to non-treated cells, while miR-451 and miR-34a were down-regulated (0.14-fold and 0.36-fold, respectively, P < 0.05). DOC treatment affected CD44+ cell frequency in MKN-45 cell line and induced significant changes in the expression of SIRT1, CXCR4, miR-21, miR-451, and miR-34a that are implicated in stemness and chemo-radioresistance, which might offer new insights for future GC therapies.

Sichong formula inhibits the proliferation and migration of human gastric cancer cells.

PurposeTraditional Chinese medicine (TCM) has gained increasing attention for the treatment of multiple chronic diseases, such as cancer. Here we aim to identify the antitumor activity of Sichong formula, a novel TCM, in human gastric cancer cells and investigate the underlying mechanisms.MethodsThe AGS and MKN45 gastric cancer cells were treated with Sichong formula at different concentrations. The proliferation rates were tested by CCK-8 and colony formation assays. Cell migration and invasion were tested by scratch and transwell assays. Gelatin zymography was used to detect the matrix metalloproteinase 9 (MMP9) activity in cell suspendents. Cell apoptosis was analyzed by Annexin V/PI staining and flow cytometry. The expression of interest proteins was tested by Western blot.ResultsCell proliferation analysis indicated that Sichong formula inhibited cell viability of AGS and MKN45 cells in a dose- and time-dependent manner. The IC50 values were 240 μg/mL and 200 μg/mL for AGS and MKN45 cells, respectively. Furthermore, we found that Sichong formula could inhibit the invasion and migration of gastric cancer cells, which might be mediated by the downregulation of MMP9 activity. Flow cytometry results indicated that Sichong formula induced apoptosis in gastric cancer cells through upregulation of Bax/Bcl2 ratio and activation of caspase cascade. The results from Western blot indicated that Sichong formula resulted in cell autophagy and inactivation of AKT signaling pathway.ConclusionOur data suggest that Sichong formula inhibits the proliferation and migration and induces apoptosis in human gastric cancer cells. The inhibitory effect of Sichong formula was, at least partly, mediated by cell autophagy and AKT pathway.

Oleanolic acid reduces aerobic glycolysis-associated proliferation by inhibiting yes-associated protein in gastric cancer cells

Gastric cancer represents a common malignancy of digestive tract with high incidence and mortality. Increasing evidence suggests that the growth of gastric tumor cells relies largely on aerobic glycolysis. Currently, many potential anti-cancer candidates are derived from natural products. Here, we evaluated the effects of oleanolic acid (OA), a triterpenoid component widely found in the plants of Oleaceae family, on aerobic glycolysis and proliferation in human MKN-45 and SGC-7901 gastric cancer cells. Our results demonstrated that OA reduced the viability and proliferation of gastric cancer cells and inhibited the expression of cyclin A and cyclin-dependent kinase 2. OA blocked glycolysis in these cells evidenced by decreases in the uptake and consumption of glucose, intracellular lactate levels and extracellular acidification rate. Glycolysis inhibitor 2-deoxy-d-glucose, similar to OA, suppressed gastric cancer cell proliferation. OA also decreased the expression and intracellular activities of glycolysis rate-limiting enzymes hexokinase 2 (HK2) and phosphofructokinase 1 (PFK1). Moreover, OA downregulated the expression of hypoxia inducible factor-1α (HIF-1α) and decreased its nuclear abundance. Upregulation of HIF-1α by deferoxamine rescued OA-inhibited HK2 and PFK1. Furthermore, OA reduced the nuclear abundance of yes-associated protein (YAP) in gastric tumor cells. YAP inhibitor verteporfin, similar to OA, downregulated the expression of HIF-1α and glycolytic enzymes in gastric cancer cells; whereas overexpression of YAP abrogated all these effects of OA. Collectively, inhibition of YAP was responsible for OA blockade of HIF-1α-mediated aerobic glycolysis and proliferation in human gastric tumor cells. OA could be developed as a promising candidate for gastric cancer treatment.