Evaluating the Magnolol Anticancer Potential in MKN-45 Gastric Cancer Cells.

Background and Objectives: Combination therapy improves the effect of chemotherapy on tumor cells. Magnolol, used in treating gastrointestinal disorders, has been shown to have anti-cancer properties. We investigated the synergistic effect of cisplatin and magnolol on the viability and maintenance of MKN-45 gastric cancer cells. Materials and Methods: The toxicity of magnolol and/or cisplatin was determined using the MTT technique. The trypan blue method was used to test magnolol and/or cisplatin's effect on MKN-45 cell growth. Crystal violet staining was used to assess the treated cells' tendency for colony formation. The expression of genes linked to apoptosis, cell cycle arrest, and cell migration was examined using the qPCR method. Results: According to MTT data, using magnolol and/or cisplatin significantly reduced cell viability. The ability of the treated cells to proliferate and form colonies was also reduced considerably. Magnolol and/or cisplatin treatment resulted in a considerable elevation in Bax expression. However, the level of Bcl2 expression was dramatically reduced. p21 and p53 expression levels were significantly increased in the treated cells, while MMP-9 expression was significantly reduced. Conclusions: These findings show that magnolol has a remarkable anti-tumor effect on MKN-45 cells. In combination with cisplatin, magnolol may be utilized to overcome cisplatin resistance in gastric cancer cells.

MIR4435-2HG in exosomes promotes gastric carcinogenesis by inducing M2 polarization in macrophages.

Gastric cancer (GC) is a cancer with a high mortality rate. lncRNAs play a role in regulating GC tumorigenesis. In this paper, we analyzed differentially expressed lncRNAs between GC and adjacent normal tissues using multiple bioinformatics tools to identify new potential targets in GC. Cell viability and migration ability were detected using the Cell Counting Kit-8 (CCK-8) and transwell assays, MIR4435-2HG was negatively correlated with the survival rate of GC patients, and by inhibiting the activity of MIR4435-2HG, the viability and migration ability of GC cells could be reduced. In addition, RT- qPCR and western blot to detect gene and protein level expression, transmission electron microscopy and nanoparticle tracking analysis (NTA) to study the efficiency of exosome isolation, and flow cytometry to observe cell differentiation were employed, delivery of MIR4435-2HG shRNA via MKN45 cell-derived exosomes significantly reversed the MKN45 exosome-induced M2 polarization in macrophages. Furthermore, the low expression of MIR4435-2HG in MKN45 cell-derived exosomes inhibited the Jagged1/Notch and JAK1/STAT3 pathways in macrophages; MIR4435-2HG downregulated exosomes were found to significantly inhibit GC tumor growth in vivo by establishing a mouse model. In short, MKN45 cell-derived exosomes deliver lncRNA MIR4435-2HG, which promotes gastric carcinogenesis by inducing macrophage M2 polarization.

[Interference of CTPS gene promotes toosendanin-induced apoptosis of human gastric cancer MKN-45 cells].

OBJECTIVE: To investigate the effect of interference of CTPS gene on toosendanin-induced apoptosis of gastric cancer MKN-45 cells. METHODS: Bioinformatic analysis was used to analyze CTPS gene expression in human gastric cancer tissues and the overall survival of gastric cancer patients with high CTPS gene expression. Human gastric cancer MKN-45 cells were transfected with a short hairpin interfering RNA targeting CTPS gene, and 48 h later, qRT-PCR and Western blotting were used to detect cellular expression CTPS at both the mRNA and protein levels. MKN-45 cells with CTPS knockdown were treated with 80 nmol/L toosendanin for 48 h, and the cell viability was assessed with MTT assay; the cell morphology was observed using laser confocal microscope, and the expression of γH2AX was detected with immunofluorescence assay. RESULTS: Bioinformatic analysis suggested that CTPS was highly expressed in human gastric cancer tissues, and gastric cancer patients with high CTPS gene expression had a shorter overall survival. MKN-45 cells transfected with Sh-CTPS interference vector showed significantly lowered cell survival rate (P < 0.01) with obvious cell shrinkage, irregular morphology, typical apoptotic changes, and increased cell apoptosis rate (P < 0.05). Treatment of the transfected cells with 80 nmol/L toosendanin for 48 h resulted in further reduction of the cell survival rate (P < 0.001), and the cells showed an increased apoptotic rate (P < 0.05) with appearance of apoptotic bodies. CONCLUSION: Interference of CTPS gene can promote TSN-induced apoptosis of gastric cancer MKN-45 cells.

Evaluation of Pharmacokinetics of Boronophenylalanine and Its Uptakes in Gastric Cancer.

Boron neutron capture therapy (BNCT), a cellular-level particle radiation therapy, combines boron compounds selectively delivered to tumor tissue with neutron irradiation. Boronophenylalanine (BPA) is a boron compound widely used in malignant melanoma, malignant brain tumors, and recurrent head and neck cancer. However, neither basic nor clinical research was reported for the treatment of gastric cancer using BPA. Selective distribution of boron in tumors rather than that in blood or normal tissue prior to neutron irradiation is required for the successful treatment of BNCT. This study evaluated the pharmacokinetics and safety of 10B-labeled BPA (10B-BPA, abbreviated as BPA) and its uptakes in gastric cancer. Pharmacokinetics and safety were evaluated in Sprague-Dawley (SD) rats intravenously injected with BPA. The uptakes of boron in gastric cancer cell line MKN45 and in cell-derived xenografts (CDX) and patient-derived xenografts (PDX) animal models were measured. The results showed that the boron concentration in the blood of rats decreased fast in the first 30 min followed by a steady decrease following the observation time, having a half-life of 44.11 ± 8.90 min and an AUC-last of 815.05 ± 62.09 min×µg/ml. The distribution of boron in different tissues (heart, liver, lung, stomach, and small intestine) of rats revealed a similar pattern in blood except for that in the brain, kidney, and bladder. In MKN45 cells, boron concentration increased in a time- and concentration-dependent manner. In both CDX and PDX animal models, the boron is preferentially distributed in tumor tissue rather than in blood or normal tissues. In addition, BPA had no significant adverse effects in rats. Taken together, the results suggested that BPA revealed a fast decrease in boron concentration in rats and is more likely to distribute in tumor cells and tissue.

The Expression and Effect of ABCE1 in Gastric Adenocarcinoma.

ATP-binding cassette E1 (ABCE1) is mainly related to the regulation of viral infection, cell multiplication, and anti-apoptosis. Previous reports confirmed the central role in the regulation of ABCE1 in liver and breast cancer; however, its potential role in gastric adenocarcinoma remains unclear. In our study, siRNA and plasmid were transfected to construct gastric cancer cell lines with low and overexpression of ABCE1, and Western blot, RT-qPCR, and immunohistochemical staining were used to detect ABCE1 expression levels in gastric cancer tissues and cell lines. The effects of ABCE1 on cell growth, metastasis, invasion, cell cycle, and drug resistance were investigated using CCK-8 test, wound healing assay, and clone formation experiment. Functional experiments indicated that si-ABCE1 decreased the proliferation, metastasis, and invasion of gastric adenocarcinoma. Meanwhile, si-ABCE1 has significantly promoted EMT process and enhanced the sensitivity of paclitaxel and cisplatin. In vivo experiments also confirmed that si-ABCE1 group had significantly smaller tumors, and immunohistochemical staining results showed the tumor growth in si-ABCE1 group was reduced obviously. In summary, we found ABCE1 is considered as a crucial role in the evolution of gastric adenocarcinoma and could be a viable therapeutic target for the disease.

Improving Tirapazamine (TPZ) to Target and Eradicate Hypoxia Tumors by Gold Nanoparticle Carriers.

Tumor hypoxia is a hallmark of solid tumors and emerged as the therapeutic target for cancer treatments, such as a prodrug Tirapazamine (TPZ) activated in hypoxia. To increase tumor accumulation, gold nanoparticles (GNPs) were selected to conjugate with TPZ. In this study, we successfully formulated and assessed the biochemical and therapeutic roles of the conjugated gold nanoparticles-Tirapazamine (GNPs-TPZ) on therapeutic assessments of MKN45-induced xenograft animal model. The results indicated that GNPs-TPZ was a potential nanomedicine for selectively targeting hypoxia tumors coupled with decreased side effects on healthy tissue or organs. TPZ significantly reduced cell viability of hypoxic gastric cancer MKN45 cells, but not in cells incubated in normoxia condition. For improving tumor targeting efficiency, furthermore, the GNPs drug carrier was conjugated to TPZ via biding mediator bovine serum albumin (BSA), and we demonstrated that this conjugated GNPs-TPZ retained the unique characteristics of hypoxic toxin and possessed the adequate feature of systemic bio-distributions in animals. GNPs-TPZ nanoparticles revealed their superior affinity to hypoxia tumors in the MKN45 xenograft. Moreover, GNPs-TPZ treatments did not significantly alter the biochemical parameters of blood samples acquired from animals. Taken together, TPZ, a prodrug activated by hypoxia, was conjugated with GNPs, whereas BSA severed as an excellent binding agent for preparing the conjugated GNPs-TPZ nanomedicines. We demonstrated that GNPs-TPZ enhanced tumor targeting, resulting in higher therapeutic efficacy compared to TPZ. We suggest that it may sever as an adjuvant treatment or combined therapy with other chemotherapeutics for the treatment of cancer patients in the future.

[Profilin 2 is highly expressed in gastric cancer and promotes tumor cell proliferation and migration].

OBJECTIVE: To investigate the expression of profilin 2 (PFN2) in gastric cancer and assess its potential value as a novel prognostic indicator and a therapeutic target. METHODS: We collected gastric cancer and paired adjacent tissues from 100 patients for immunohistochemical detection of PFN2 expression. According to the expression level of PFN2, the patients were divided into two groups with high (46 cases) and low (48 cases) PNF2 expression in cancer tissues, and also into two groups with high (26 cases) and low (49 cases) PNF2 expression in adjacent tissues. Chi-square test, Spearman correlation and KaplanMeier survival analysis were used to analyze the relationship between PFN2 protein expression level and the patients' clinical parameters. We also tested the effects of PFN2 knockdown and overexpression on the proliferation and migration of MKN-45 cells using Transwell assay and CCK-8 assay. RESULTS: The expression of PFN2 protein was significantly higher in gastric cancer tissues than in adjacent tissues (P < 0.01). PFN2 expression was positively correlated with M-stage of gastric cancer and VEGFR expression in the tumor tissues (P < 0.01). A high expression of PFN2 protein was significantly correlated with a poor prognosis of gastric cancer patients (P < 0.01), and was an independent predictor of the prognosis of gastric cancer. In MKN-45 cells, the cells overexpressing PFN2 showed significantly stronger proliferation and migration abilities than those with PFN2 knockdown (P < 0.001). CONCLUSION: PFN2 protein is highly expressed in gastric cancer tissues to promote the proliferation and migration of the tumor cells. PFN2 may serve as a potential diagnostic marker, a prognostic indicator and a therapeutic target for gastric cancer.

A Decrease in CD44 on Cell Surfaces (MKN-45 cell line) After RELA Knockout Using CRISPR/Cas9.

The NF-kB signaling pathway was introduced as a key pathway in carcinogenesis that is induced by inflammation in gastrointestinal malignancies. The RelA transcription factor is an important component of this signaling pathway. Furthermore, CD44 is implicated in the tumorigenesis and metastasis of gastric cancer. The aim of this study was to assay the effect of RELA knockout on CD44 expression in MKN45 cells. CRISPR/Cas9 was used to knock out RELA in MKN-45. The median fluorescence intensity (MFI) of CD44 before and after RELA knockout is analyzed in MKN45. The CRISPR/Cas9 vector pSpCas9 (BB)-2A-Puro (PX459) was used for gRNA cloning (two guides). The MKN-45 cell line was co-transfected. The purified co-transfected cells with puromycin were cultured and used for the RELA gene expression assay by real-time PCR. Flow cytometry was used for the analysis of the MFI of CD44+ in MKN45. The results showed that 180 nucleotide sequences between exon 2 and exon 3 of RELA were deleted in MKN45. RELA expression significantly (P<0.001) decreased after CRISPR/Cas9 knockout. Compared to the control group, the MFI of CD44 in transfected cells significantly decreased (P <0.001). Knockout of RELA significantly decreased CD44 expression in MKN45 cells. It can be concluded that the NF-kB signaling pathway via RELA is related to CD44 expression and consequently the tumorigenesis of gastric cancer. More studies about this relationship are recommended.

Effect of NAIF1 on IFIT Family Proteins in Gastric Cancer / 肿瘤防治研究

Objective To investigate the effect of NAIF1 in gastric cancer cell lines MKN45. Methods We constructed pLVX-Tight-Flag-NAIF1-puro plasmid with Tet-on system. DOX was added to induce NAIF1 expression in MKN45 cells. The cells were collected at 0, 6, 12 and 24 hours after DOX addition for gene expression microarray detection and biological analysis of differentially expressed genes. qRT-PCR and Western blot were used to verify the changes in mRNA and protein levels of the selected target differential genes. Results The biological analysis of gene microarray hybridization results showed that IFIT1, IFIT2 and IFIT3 expression significantly increased at 24h, qRT-PCR also showed this change, and Western blot further verified the change in protein level. However, IFIT5 showed no significant change in mRNA and gene expression. Conclusion Over-expression of NAIF1 in gastric cancer cells can promote the expression of some immune system-related IFIT family proteins.

Profilin 2 is highly expressed in gastric cancer and promotes tumor cell proliferation and migration / 南方医科大学学报

OBJECTIVE@#To investigate the expression of profilin 2 (PFN2) in gastric cancer and assess its potential value as a novel prognostic indicator and a therapeutic target.@*METHODS@#We collected gastric cancer and paired adjacent tissues from 100 patients for immunohistochemical detection of PFN2 expression. According to the expression level of PFN2, the patients were divided into two groups with high (46 cases) and low (48 cases) PNF2 expression in cancer tissues, and also into two groups with high (26 cases) and low (49 cases) PNF2 expression in adjacent tissues. Chi-square test, Spearman correlation and KaplanMeier survival analysis were used to analyze the relationship between PFN2 protein expression level and the patients' clinical parameters. We also tested the effects of PFN2 knockdown and overexpression on the proliferation and migration of MKN-45 cells using Transwell assay and CCK-8 assay.@*RESULTS@#The expression of PFN2 protein was significantly higher in gastric cancer tissues than in adjacent tissues (P < 0.01). PFN2 expression was positively correlated with M-stage of gastric cancer and VEGFR expression in the tumor tissues (P < 0.01). A high expression of PFN2 protein was significantly correlated with a poor prognosis of gastric cancer patients (P < 0.01), and was an independent predictor of the prognosis of gastric cancer. In MKN-45 cells, the cells overexpressing PFN2 showed significantly stronger proliferation and migration abilities than those with PFN2 knockdown (P < 0.001).@*CONCLUSION@#PFN2 protein is highly expressed in gastric cancer tissues to promote the proliferation and migration of the tumor cells. PFN2 may serve as a potential diagnostic marker, a prognostic indicator and a therapeutic target for gastric cancer.

miR-875-5p inhibits the proliferation, migration and invasion of gastric cancer cells by targeting USF2 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miR-875-5p对胃癌细胞增殖、迁移和侵袭的影响及其机制。方法：采用qPCR法检测胃癌细胞BGC-823、HGC-27、MGC-803、SGC-7901、AGS、MKN-45 和胃黏膜上皮细胞GES-1中miR-875-5p的表达水平。利用脂质体转染技术，分别将miR-875-5p模拟物/抑制剂（mimic/inhibitor）及其阴性对照质粒（miR-NC/Anti-miR-NC）转染至AGS细胞/MKN-45细胞，构建过表达/抑制miR-875-5p的细胞模型，空白对照组（Control组）不转染。通过CCK-8、克隆形成、Transwell等实验分别检测miR-875-5p表达变化对细胞增殖、克隆形成、迁移和侵袭的影响。采用双荧光素酶报告基因实验验证miR-875-5p与上游刺激因子2（USF2）的靶向关系，WB实验验证miR-875-5p对USF2的调控作用并检测USF2蛋白的表达。构建MKN-45细胞裸鼠移植瘤模型，验证miR-875-5p过表达对MKN-45细胞成瘤能力的影响。结果：miR-875-5p在6种胃癌细胞中表达水平显著低于胃黏膜上皮细胞GES-1（均P<0.01）。与Control组和miR-NC组相比，miR-875-5p mimic组AGS细胞的增殖、克隆形成率、迁移和侵袭细胞数，以及USF2蛋白的表达均显著降低（P<0.05或P<0.01）；miR-875-5p inhibitor组MKN-45细胞的增殖、克隆形成率、迁移和侵袭细胞数，以及USF2蛋白的表达均显著提高（P<0.05或P<0.01）。双荧光素酶报告基因实验证明，miR-875-5p能够直接靶向USF2基因。体内成瘤实验结果表明，过表达miR-875-5p显著抑制MKN-45细胞移植瘤的生长（均P<0.01）。结论：miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭。

Therapeutic efficacy of a paclitaxel-loaded nanofibrillated bacterial cellulose (PTX/NFBC) formulation in a peritoneally disseminated gastric cancer xenograft model.

Nano-fibrillated bacterial cellulose (NFBC) is a safe, biocompatible material that can be prepared by culturing a cellulose-producing bacterium in a culture supplemented with carboxymethylcellulose (CMC) or hydroxypropylcellulose (HPC). CM-NFBC and HP-NFBC, prepared using CMC or HPC, show hydrophilicity and amphiphilicity, respectively, and thus they could be useful carriers for hydrophobic anticancer agents such as paclitaxel (PTX). In the present study, we prepared novel PTX formulations for intraperitoneal administration by associating PTX with either CM-NFBC or HP-NFBC and studied their therapeutic efficacy on peritoneally disseminated gastric cancer in a xenograft nude mouse model. Freeze-dried PTX formulations (PTX/CM-NFBC and PTX/HP-NFBC) were quickly reconstituted with saline without any foaming, compared to nanoparticle albumin-bound PTX (nab-PTX, Abraxane®). Both PTX/NFBC formulations extended the mean survival times in our xenograft murine models compared with either free PTX or nab-PTX. The PTX/NFBC formulations reduced systemic side effects of free PTX relating to weight loss. In our disseminated gastric peritoneal cancer model, the PTX/NFBC formulation increased the therapeutic index for PTX by increasing the therapeutic efficacy and decreasing toxicity. NFBCs should receive consideration as improved carriers for the clinical delivery of hydrophobic anticancer drugs such as PTX in malignancies in the abdominal cavity with peritoneal metastasis and dissemination.

Downregulation of CDH11 Promotes Metastasis and Resistance to Paclitaxel in Gastric Cancer Cells.

Background: Gastric cancer (GC) with peritoneal metastasis has an extremely poor prognosis. Paclitaxel (PTX) intraperitoneal infusion provides an effective treatment for these patients. However, GC patients with peritoneal metastasis who receiving PTX treatments tend to occur PTX-resistance accompany with more aggressive ascites and metastasis. How does this happen is still unknown. Here, we aimed to explore the mechanisms that mediate PTX-resistance and metastasis in GC with peritoneal metastasis. Methods: Ascites samples were collected before PTX infusion and after the relapse in 3 GC patients. To determine the expression of significantly changed proteins, we performed tandem mass tag (TMT) quantitative proteomics. Immunohistochemistry (IHC) staining and western blot were performed to confirm the expression of CDH11 in the PTX-resistant tissues and MKN45P-PR cells. Invasion and migration of GC cells were examined by in vitro transwell and wound healing assays and in vivo dissemination experiments. Results: CDH11 expression was downregulated in the relapsed PTX-resistant ascites, tissues and the PTX-resistant cell line MKN45P-PR. Inhibition of CDH11 expression promoted the invasion, migration and PTX resistance of MKN45P cells, while overexpression of CDH11 repressed these biological functions. Moreover, tumors disseminated in the mice peritoneal cavity induced by MKN45P-PR cells and shCDH11 cells displayed higher metastatic ability and resistance to PTX treatment. Conclusions: Our results reveal that CDH11 is inhibited in the relapsed PTX-resistant patients and the downregulated CDH11 expression promotes GC cell invasion, migration and PTX resistance. CDH11 may have the potential to serve as a predictable marker for the occurrence of PTX resistance in GC patients with peritoneal metastasis.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Promotive effect of Talin-1 protein on gastric cancer progression through PTK2-PXN-VCL-E-Cadherin-CAPN2-MAPK1 signaling axis.

OBJECTIVE: Our research group was aim to explore the molecular mechanism of Talin-1 protein affecting gastric cancer progression through PTK2-PXN-VCL-E-Cadherin-CAPN2-MAPK1 signal axis. METHODS: 12 cases of patients with gastric cancer in this hospital from 2018 to 2019 were collected. Immunohistochemistry assay and Western blotting were used to detect the expression of Talin-1, PXN, E-Cadherin, CAPN2, MAPK1 protein in gastric cancer tissue. Cell migration and invasion were measured by Transwell. RESULTS: The results showed that the expression levels of protein Talin-1, PXN and MAPK1 in gastric cancer tissues were significantly higher than that in normal tissue. The number of cell adhesion in the model group was significantly lower than that in the normal group. However, the cell adhesion number in ov-TLN1 was the highest. Transwell results showed that TLN1 could accelerate the migration and invasion abilities of gastric cancer MKN-45 cells. Moreover, Western blotting showed that protein Talin-1, PXN, E-Cadherin, CAPN2, MAPK1 in model group all increased compared with normal group. CONCLUSION: It indicated that talin-1 protein influenced the development of gastric cancer through PTK2-PXN-VCL-E-Cadherin-CAPN2-MAPK1 signal axis.

The preparation of a cold-water soluble polysaccharide from Grifola frondosa and its inhibitory effects on MKN-45 cells.

In this study, a novel water soluble polysaccharide (named GFP-4) was extracted from Grifola frondosa at 4 oC, and its preliminary structure and inhibitory effects on human gastric carcinoma MKN-45 cells through the Fas/FasL death receptor apoptosis pathway were investigated. High-performance gel permeation chromatography (HPGPC), fourier-transform infrared spectroscopy (FT-IR), and ion chromatography (IC) results showed that GFP-4 was a 1.09 × 106 Da neutral hetero polysaccharide with pyranose rings, and α- and ß-type glycosidic linkages that contained galactose, glucose, and mannose at a molar ratio of 1.00:3.45:1.19. MTT results indicated that GFP-4 significantly inhibited the proliferation of MKN-45 cells in a concentration-dependent manner. The H&E staining and Hoechst 33342/PI double staining results showed that GFP-4-treated MKN-45 cells were subjected to underwent typical apoptotic morphologic changes such as nuclear pyknosis, chromatin condensation, and an increase of membrane permeability. Annexin V-FITC/PI double staining, cell cycle analysis, and western blot results revealed the GFP-4 induced MKN-45 cells apoptosis through the Fas/FasL-mediated death receptor pathway with cells arrested at the G0/G1 phase. These data indicate that GFP-4 is a promising candidate for treating gastric cancer and provide a theoretical basis for the future development and utilization of G. frondosa clinically.

Frequency of CD44 positive cells in MKN45 cell line after treatment with docetaxel in two and three-dimensional cell cultures.

Three-dimensional (3D) cell culture is more similar to in vivo studies and suitable for studies of interactions between cells and extracellular matrix. CD44 is a cell surface receptor that can relate with the extracellular matrix molecules. CD44 in gastric cancer (GC) is a metastatic and drug resistance marker. In this study the quantity of CD44+ cells in MKN-45 cell line in response to half maximal inhibitory concentration (IC50) dose of Docetaxel (DOC) was measured in 2D and 3D cultures. MKN-45 cell line was cultured in 2D and 3D environments. For 3D culture, rat gastric tissue was separated and decellularized and MKN-45 cells were injected and cultured in the prepared matrix. The frequency of CD44+ cells in 2D and 3D cultures were analyzed before and after treatment with IC50 of DOC by flow cytometry and immunohistochemistry. Despite different environmental conditions, The frequency of CD44+ cells increased significantly in 2D and 3D environments after treatment with IC50 of DOC (P < 0.05). Given the advantages of 3D, this environment seems more appropriate for study about CD44+ cells and drug resistance in GC.

A selective and sensitive turn-on chemosensor for detection of Fe3+ in aqueous solution and its cell imaging in dorsal root ganglia neurons and MKN-45 cells.

A new turn-on fluorescent chemosensor (RBTM) for Fe3+ was designed based on Rhodamine B and a thiocarbonylimidazole moiety. The spectroscopic probe used for characterization of the synthesized system showed 300-fold fluorescence enhancement for the detection of Fe3+ with a 1:1 stoichiometry in EtOH/H2O solution (2:1, v/v, HEPES buffer, 1 mM, pH 7.30). Upon addition of Fe3+ in aqueous ethanol, the probe displayed a significant fluorescence enhancement and a distinct color change (colorless to pink) that can be detected by the naked eye. The binding constant between the probe and Fe3+ was determined to be 1.16 × 104 M-1 and the corresponding detection limit was calculated to be 0.256 µM. In addition, the energy gaps between the HOMO and LUMO in RBTM and RBTM-Fe3+ were calculated using DFT calculations to be 92.93 kcal/mol and 37.49 kcal/mol, respectively. The results indicate that binding of Fe3+ to RBTM lowered the HOMO-LUMO energy gap of the complex and stabilized the system. Fluorescence imaging experiments demonstrated that RBTM can be used as a fluorescent probe to detect Fe3+ in MKN-45 cells and dorsal root ganglia, thus revealing that RBTM could be used for biological applications.

[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.

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

PURPOSE: 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). METHODS: 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. RESULTS: The half-maximal inhibitory concentration (IC50) of DOC was 10 µg/ml after 48 h. 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). CONCLUSION: 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.