[Effect of Circular RNA hsa_circ_0067582 on the Proliferation and Invasion Ability of Gastric Cancer Cells].

Objective To investigate the effects on cell proliferation and invasion of the circular RNA hsa_circ_0067582 in gastric cancer(GC). Methods After hsa_circ_0067582 overexpression (Oe-circ_0067582) plasmid was transfected into AGS and SGC-7901 cells,the cell viability,proliferation,invasion ability,and apoptosis were detected by CCK-8,colony formation and EdU assays,Transwell assay,and flow cytometry,respectively.Western blotting was employed to detect the expression levels of proteins related to the cell apoptosis and epithelial-mesenchymal transition(EMT).The effect of Oe-circ_0067582 on the growth of SGC-7901 cells in nude mice was observed.Bioinformatics tools were used to predict the binding target miRNA of hsa_circ_0067582,and the competing endogenous RNA(ceRNA)regulatory network was established.Finally,functional enrichment was performed to analyze the biological functions of the target genes of the predicted miRNA. Results Compared with the pLO-ciR(empty plasmid)group,the Oe-circ_0067582 group in AGS and SGC-7901 cells attenuated the cell viability(t=7.883,P=0.001;t=5.679,P=0.005),proliferation(t=6.709,P=0.003;t=5.857,P=0.003),and invasion ability(t=7.782,P=0.002;t=6.342,P=0.003)and induced cell apoptosis(t=7.225,P=0.002;t=11.509,P=0.001).Western blotting showed that the Oe-circ_0067582 group in AGS and SGC-7901 cells up-regulated the protein levels of cysteinyl aspartate specific proteinase (Caspase) 3(t=6.863,P=0.002;t=7.024,P=0.001),Caspase 7(t=3.295,P=0.04;t=6.008,P=0.004),Caspase 9(t=4.408,P=0.012;t=6.278,P=0.004),and E-cadherin(t=12.453,P=0.002;t=10.867,P=0.001),while down-regulated those of Vimentin(t=7.242,P=0.002;t=5.694,P=0.004)and N-cadherin(t=6.480,P=0.003;t=7.446,P=0.001).Furthermore,Oe-circ_0067582 significantly inhibited the growth of tumor in the SGC-7901 tumor-bearing nude mice(t=3.526,P=0.017).The prediction based on TargetScan and miRnada suggested that hsa_circ_0067582 can competitively bind to hsa-miR-181b-3p,hsa-miR-337-3p,hsa-miR-421,and hsa-miR-548d-3p.The functional enrichment indicated that the target genes of miRNA were involved in multiple cancer-related biological processes including negative regulation of apoptotic process,gene expression,transcriptional misregulation in cancer,transforming growth factor-ß,and p53 signaling pathways. Conclusion Oe-circ_0067582 can inhibit the proliferation and attenuate EMT process to reduce the invasion ability of AGS and SGC-7901 cells,which provides a new target for the treatment of GC.

3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1,2,4] triazolo [3,4-b][1,3,4] thiadiazine, a novel tubulin inhibitor, evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells.

Gastric cancer and cervical cancer are two major malignant tumors that threaten human health. The novel chemotherapeutic drugs are needed urgently to treat gastric cancer and cervical cancer with high anticancer activity and metabolic stability. Previously we have reported the synthesis, characterization and identification of a novel combretastatin A-4 analog, 3-(3-methoxyphenyl)-6-(3-amino-4- methoxyphenyl) -7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazine (XSD-7). In this study, we sought to investigate its anticancer mechanisms in a human gastric cancer cell line (SGC-7901 cells) and human cervical carcinoma cell line (HeLa cells). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that XSD-7 induced cytotoxicity in SGC-7901 and HeLa cells with inhibitory concentration 50 values of 0.11 ± 0.03 and 0.12 ± 0.05 µM, respectively. Immunofluorescence studies proved that XSD-7 inhibited microtubule polymerization during cell division in SGC-7901 and HeLa cells. Then, these cells were arrested at G2/M cell cycle and subsequently progressed into apoptosis. In further study, mitochondrial membrane potential analysis and Western blot analysis demonstrated that XSD-7 treatment-induced SGC-7901 cell apoptosis via both the mitochondria-mediated pathway and the death receptor-mediated pathway. In contrast, XSD-7 induced apoptosis in HeLa cells mainly via the mitochondria-mediated pathway. Hence, our data indicate that XSD-7 exerted antiproliferative activity by disrupting microtubule dynamics, leading to cell cycle arrest, and eventually inducing cell apoptosis. XSD-7 with novel structure has the potential to be developed for therapeutic treatment of gastric cancer and cervical cancer.

The synergistic anti-tumor effect of schisandrin B and apatinib.

The synergistic anti-tumor effect of schisandrin B (Sch.B) and apatinib was investigated in vitro. The CCK-8 assay revealed that Sch.B enhanced the inhibition of apatinib on cell proliferation by arresting cell cycle in G0/G1 phase. Sch.B also potentiated the suppression of apatinib on cell migration and invasion, by means of wound-healing and transwell invasion assay. Flow cytometry results showed that Sch.B enhanced apoptosis induced by apatinib. The results were confirmed by western blot analysis of the proteins MMP-9, and Bax caspase-9, and -12. These results suggest that combining apatinib and Sch.B is an effective therapeutic strategy for preventing GC progression. [Formula: see text].

Activation of LXRß inhibits proliferation, promotes apoptosis, and increases chemosensitivity of gastric cancer cells by upregulating the expression of ATF4.

Recently, great advances have been achieved in both surgery and chemotherapy for the treatment of gastric cancer, but there is still poor prognosis for this disease. The aim of this study is to investigate the role of liver X receptor ß (LXRß) in chemosensitivity of gastric cancer SGC7901 cells. From 171 patients with gastric cancer, the gastric cancer and paracancerous tissues were selected to measure the expression of LXRß and ATF4. Gastric cancer cell lines were cultured and screened to figure out the proliferation and apoptosis of gastric cancer SGC7901 cells with the treatment of LXRß agonist (GW3965), ATF4 short hairpin RNA (shRNA), and chemotherapy drug paclitaxel. The expression of apoptosis-related gene cleaved caspase-3 was detected by Western blot analysis. First, we found that the expressions of LXRß and ATF4 in gastric cancer tissues and cells were significantly lower than those in their paracancerous tissues and gastric mucosal epithelial cells. In addition, activation of LXRß and paclitaxel treatment suppressed proliferation of SGC7901 cells, and the expression of ATF4 was upregulated in a concentration-dependent manner. Furthermore, shRNA significantly inhibited the expression of ATF4 and blocked the chemosensitivity of SGC7901 cells to LXRß activation. Our study demonstrates that the expression of LXRß was low in gastric cancer. In addition, activation of LXRß may inhibit the proliferation of gastric cancer cells, promote apoptosis, and increase chemosensitivity by upregulating the expression of ATF4.

Discovery and anticancer evaluation of a formononetin derivative against gastric cancer SGC7901 cells.

Background Gastric cancer (GC) is the second most common cause of cancer-related death worldwide. Novel anticancer drugs against gastric cancer are urgently needed. Methods Compound 10 was designed and synthesized via a molecular hybridization strategy based on the natural product formononetin. It was evaluated for their antiproliferative activity against three gastric cancer cell lines (SGC7901, MKN45 and MGC803). Results Derivative 10 displayed potently antiproliferative activity with an IC50 value of 1.07 µM against SGC7901 cells. Derivative 10 could inhibit the growth and migration against gastric cancer SGC7901 cells through the Wnt/ß-Catenin and AKT/mTOR pathways. From the in vivo expremints, it could effectively inhibited SGC7901 xenograft tumor growth in vivo without significant loss of the body weight. Conclusion Derivative 10 is an novel antitumor agent with potential for further clinical applications to treat gastric cancer. Graphical abstract.

Gramicidin inhibits human gastric cancer cell proliferation, cell cycle and induced apoptosis.

BACKGROUND: Gastric cancer is a common malignant tumor with high morbidity and mortality worldwide, which seriously affects human health. Gramicidin is a short peptide antibiotic which could be used for treating infection induced by bacteria or fungi. However, the anti-cancer effect of gramicidin on gastric cancer cells and its underlying mechanism remains largely unknown. RESULTS: Gastric cancer cells SGC-7901, BGC-823 and normal gastric mucosal cells GES-1 were treated with different concentrations of gramicidin respectively. The results of CCK-8 experiment revealed cellular toxicity of gramicidin to cancer cells while cell colony formation assay showed that gramicidin significantly inhibited the proliferation of gastric cancer cells, but had little effect on normal gastric mucosal cells. In addition, the wound healing assay showed that gramicidin inhibited the migration of SGC-7901 cell. Meanwhile, apoptosis and cell cycle analysis revealed that gramicidin induced cell apoptosis with G2/M cell cycle inhibition. Furthermore, western blot analysis demonstrated that gramicidin down-regulated the expression of cyclinD1 and Bcl-2 as well as the FoxO1 phosphorylation. CONCLUSIONS: The current study illustrated the anti-tumor activity of gramicidin on gastric cancer cells, providing a possibility for gramicidin to be applied in clinical practice for the treatment of gastric cancer.

[Mechanism of ß-carboline alkaloids inhibiting migration and invasion of SGC-7901 cells].

To explore the mechanism of ß-carboline alkaloids inhibiting the migration and invasion of SGC-7901 cells and its correlation with FAK gene expression,CCK-8 method was used to determine the inhibitory rate of ß-carboline alkaloids on the proliferation of gastric cancer SGC-7901 cells under different concentrations.The effect of ß-carboline alkaloids on the migration and invasion of SGC-7901 cells was used by Transwell compartment.Detection of mRNA and protein expression of FAK genes were used by qRT-PCR and Western blot.Then si-FAK-1051 recombinant plasmid was transfected into SGC-7901 cells.FAK gene silencing effect was identified by qRT-PCR and Western blot technique again.Finally,the effects of FAK gene silencing on proliferation and migration of gastric cancer SGC-7901 cells were detected by CCK-8 kit and Transwell chamber assay respectively.With the increase of the concentration ofß-carboline alkaloids,the inhibitory rate of SGC-7901 cells in human gastric cancer cells increased gradually,with IC5013.364 mg·L-1.The number of SGC-7901 cells of Transwell compartment in the positive experimental group(5-FU,5 mg·L-1) and the ß-carboline alkaloids group decreased significantly(P<0.01) and the number of SGC-7901 cells in the ß-carboline alkaloids group was significantly lower than that in the positive experimental group(P<0.01).Compared with the blank control group,the mRNA and protein expression level of FAK genes in the positive experimental group was significantly lower than that in the experimental group of ß-carboline alkaloids(P<0.05).After transfection of si-FAK-1051 into gastric cancer SGC-7901 cells,the expression of mRNA and protein of FAK gene was significantly down regulated(P<0.05).SGC-7901 cell proliferation and cell migration ability also decreased significantly(P<0.05).ß-carboline alkaloids are more effective than 5-FU in inhibiting migration and invasion of gastric cancer SGC-7901 cells,and the mechanism may be related to the inhibition of mRNA and protein expression of FAK gene by ß-carboline alkaloids.

Effects of fucoxanthin on autophagy and apoptosis in SGC-7901cells and the mechanism.

Autophagy and apoptosis are involved in the development of a variety of cancers. Fucoxanthin is a natural compound known to have antitumor effects, so we aimed to explore its effects on autophagy and apoptosis in gastric cancer SGC7901 cells. Specifically, we performed methyl thiazolyl tetrazolium assay, transmission electron microscopy, real-time polymerase chain reaction, Western blot analysis, immunofluorescence assay, and cell apoptosis analysis to clarify the role of fucoxanthin in SGC-7901 cells. Our results indicate that fucoxanthin significantly inhibits the viability of SGC-7901 cells, effectively inducing both autophagy and apoptosis by up-regulating the expressions of beclin-1, LC3, and cleaved caspase-3 (CC3), and by down regulating Bcl-2. Fucoxanthin-induced autophagy also seems to occur before, and may promote apoptosis.

siRNA Targeting of the SNCG Gene Inhibits the Growth of Gastric Carcinoma SGC7901 Cells in vitro and in vivo by Downregulating the Phosphorylation of AKT/ERK.

The aim of the study was to evaluate the effects of synuclein-γ (SNCG) silencing on gastric cancer SGC7901 cells and to elucidate the associated mechanisms. pGCSIL-lentiviral siRNA targeting of the SNCG gene was employed to inhibit SNCG expression. Several experiments such as quantitative real-time PCR, Western blotting, MTT, colony formation, migration assay, and flow cytometry were performed to investigate the biological behavior of infected SGC7901 cells. BALB/c nude mice were used as tumor xenograft models to assess the effects of SNCG silencing on tumor growth. Western blot analysis was carried out to determine the relative levels of AKT, p-AKT, ERK, and p-ERK expression. Our results showed that SNCG was overexpressed in SGC7901 cells as compared to normal gastric mucosal epithelial cells. SGC7901 cells transfected with SNCG siRNA demonstrated significantly decreased gastric cancer growth (p < 0.01), reduced cell migration, cell cycle arrest in the G0/G1 phase, promoted tumor cell apoptosis (p < 0.01), and inhibited tumorigenesis in xenograft animal models. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA group than in the control groups. The results of the present study suggest that SNCG siRNA plays a significant role in the proliferation, migration, and tumorigenesis of gastric cancer by downregulating the phosphorylation of AKT and ERK. RNA interference-mediated silencing of SNCG may provide an opportunity to develop a novel treatment strategy for gastric cancer.

[Effects of orchinol on invasion, migration and Wnt3a/ß-catenin signaling pathway of human gastric cancer SGC-7901 cells].

The purpose of this study was to investigate the inhibitory effect of the main 9,10-dihydrophenanthrene orchinol isolated from Spiranthes sinensis Radix et Herba on the invasion and migration of human gastric cancer SGC-7901 cells and its preliminary molecular mechanism. SGC-7901 cells were cultured in vitro, after the cells were treated with different final concentrations(5, 10, 20, 40, 80 µmol·L⁻¹) of orchinol for 24, 48 or 72 hours, the effect of orchinol on cell viability was measured by MTT assay. Wound healing and Transwell assays were performed to determine the effects of different final concentrations(5, 10, 20, 40 µmol·L⁻¹) of orchinol for 48 hour on invasion and migration abilities of SGC-7901 cells, respectively. The protein expression levels of ß-catenin, Wnt-3α, DvL2, cyclinD1 and GSK-3ß were detected by Western blot. The results showed that 5-80 µmol·L⁻¹ orchinol inhibited the viability of SGC-7901 cells in a dose-dependent and time-dependent manner, and the IC58 values of 24, 48 and 72 hours were 77.79, 42.96 and 7.85 µmol·L⁻¹, respectively. Compared with the control group, the ability of invasion and migration of SGC-7901 cells was significantly inhibited after treated with 5, 10 and 20 µmol·L⁻¹ orchinol for 48 hours (P<0.05, P<0.01), and the dose-effect relationship was observed. The results of Western blot showed that orchinol could significantly down-regulate the protein expression levels of ß-catenin, Wnt3a, DvL2 and cyclinD1, and up-regulate the protein expression level of GSK-3ß(P<0.05, P<0.01, P<0.001). The above results suggest that orchinol can obviously inhibit the invasion and migration of SGC-7901 cells, which may be related to its inhibition of Wnt3a/ß-catenin signaling pathway and the proteins expression of downstream genes.

Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway.

BACKGROUND/AIMS: Mesenchymal stem/stromal cells (MSCs) are known to home to sites of tumor microenvironments where they participate in the formation of the tumor microenvironment and to interplay with tumor cells. However, the potential functional effects of MSCs on tumor cell growth are controversial. Here, we, from the view of bone marrow MSC-derived exosomes, study the molecular mechanism of MSCs on the growth of human osteosarcoma and human gastric cancer cells. METHODS: MSCs derived from human bone marrow (hBMSCs) were isolated and cultured in complete DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin, cell culture supernatants containing exosomes were harvested and exosome purification was performed by ultracentrifugation. Osteosarcoma (MG63) and gastric cancer (SGC7901) cells, respectively, were treated with hBMSC-derived exosomes in the presence or absence of a small molecule inhibitor of Hedgehog pathway. Cell viability was measured by transwell invasion assay, scratch migration assay and CCK-8 test. The expression of the signaling molecules Smoothened, Patched-1, Gli1 and the ligand Shh were tested by western blot and RT-PCR. RESULTS: In this study, we found that hBMSC-derived exosomes promoted MG63 and SGC7901 cell growth through the activation of Hedgehog signaling pathway. Inhibition of Hedgehog signaling pathway significantly suppressed the process of hBMSC-derived exosomes on tumor growth. CONCLUSION: Our findings demonstrated the new roles of hedgehog signaling pathway in the hBMSCs-derived exosomes induced tumor progression.

MicroRNA-1284 inhibits proliferation and induces apoptosis in SGC-7901 human gastric cancer cells.

OBJECTIVES: To explore the functional effects of miR-1284 on gastric cancer cells. RESULTS: Overexpression of miR-1284 significantly reduced SGC-7901 cell proliferation, but improved apoptosis. However, miR-1284 suppression displayed the inversed impacts. Furthermore, the protein levels of p27, Bax, procaspase-3 and active caspase-3 were up-regulated by miR-1284 overexpression, but were down-regulated by miR-1284 suppression. The level of Bcl-2 was down-regulated by miR-1284 overexpression, while it was up-regulated by miR-1284 suppression. The level of p21 was unaffected. CONCLUSION: These results suggest that miR-1284 overexpression might be a suppressor for gastric cancer via controlling of cell proliferation and apoptosis.

Platinum-zoledronate complex blocks gastric cancer cell proliferation by inducing cell cycle arrest and apoptosis.

A series of novel dinuclear platinum complexes based on the bisphosphonate ligands have been synthesized and characterized in our recent study. For the purpose of discovering the pharmacology and action mechanisms of this kind of compounds, the most potent compound [Pt(en)]2ZL was selected for systematic investigation. In the present study, the inhibition effect on the human gastric cancer cell lines SGC7901 and action mechanism of [Pt(en)]2ZL were investigated. The traditional 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and colony formation assay were carried out to study the effect of [Pt(en)]2ZL on the cell viability and proliferation capacity, respectively. The senescence-associated ß-galactosidase staining and immunofluorescence staining were also performed to assess the cell senescence and microtubule polymerization. Fluorescence staining and flow cytometry (FCM) were used to monitor the cell cycle distribution and apoptosis, and Western blot analysis was applied to examine the expression of several apoptosis-related proteins. The results demonstrated that [Pt(en)]2ZL exhibited remarkable cytotoxicity and anti-proliferative effects on the SGC7901 cells in a dose- and time-dependent manner, and it also induced cell senescence and abnormal microtubule assembly. The cell apoptosis and cell cycle arrest induced by [Pt(en)]2ZL were also observed with the fluorescence staining and FCM. The expressions of cell cycle regulators (p53, p21, cyclin D1, cyclin E, and cyclin-dependent kinase (CDK)2) and apoptosis-related proteins (Bcl-2, Bax, caspase-3, poly ADP ribose polymerase (PARP), and survivin) were regulated by the treatment of [Pt(en)]2ZL, resulting in the cell cycle arrest and apoptosis. Therefore, [Pt(en)]2ZL exerted anti-tumor effect on the gastric cancer via inducing cell cycle arrest at G1/S phase and apoptosis.

Organic two-photon nanoparticles modulate reactive oxygen species, intracellular calcium concentration, and mitochondrial membrane potential during apoptosis of human gastric carcinoma SGC-7901 cells.

OBJECTIVES: To investigate the biocompatibility of human gastric carcinoma cells (SGC-7901) with organic two-photon nanoparticles (NPs). RESULTS: Different concentrations of NPs were incubated with SGC-7901 cells for different times. The levels of cell apoptosis, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential (MMP) were measured by staining the SGC-7901 cells with Annexin V-FITC/PI, 2',7'-dichlorofluorescin diacetate, Fluo-3 AM, and Rhodamine 123, followed by the flow cytometry assay. NPs at <4 µg/ml, did not have any significant effect on apoptosis, necrosis, generation of ROS, increase of intracellular Ca(2+) concentration or decrease of MMP in SGC-7901 cells, but >4 µg/ml had a major effects on all the above mentioned parameters. CONCLUSION: 2,5,2',5'-Tetra(4-N,N-diphenylamine styryl) biphenyl NPs can be used at an appropriate concentration as a safe drug carrier or imaging marker and may serve as an effective tool for developing a photodynamic cancer therapy.

Synthesis, characterization and theoretical calculations of (1,2-diaminocyclohexane)(1,3-diaminopropane)gold(III) chloride complexes: in vitro cytotoxic evaluations against human cancer cell lines.

The gold(III) complexes of the type (1,2-diaminocyclohexane)(1,3-diaminopropane)gold(III) chloride, [(DACH)Au(pn)]Cl3, [where DACH = cis-, trans-1,2- and S,S-1,2-diaminocyclohexane and pn = 1,3-diaminopropane] have been synthesized and characterized using various spectroscopic and analytical techniques including elemental analysis, UV-Vis and FTIR spectroscopy; solution as well as solid-state NMR measurements. The solid-state (13)C NMR shows that 1,2-diaminocyclohexane (1,2-DACH) and 1,3-diaminopropane (pn) are strongly bound to the gold(III) center via N donor atoms. The stability of the mixed diamine ligand gold(III) was checked by UV-Vis spectroscopy and NMR measurements. The molecular structure of compound 1 (containing cis-1,2-DACH) was determined by X-ray diffraction analysis. The structure of 1 consists of [(cis-DACH)Au(pn)](3+) complex ion and chloride counter ions. Each gold atom in the complex ion adopts a distorted square-planar geometry. The structural details and relative stabilities of the four possible isomers of the complexes were also estimated at the B3LYP/LANL2DZ level of theoretical calculations. The computational study demonstrates that trans- conformations are slightly more stable than the cis- conformations. The antiproliferative effects and cytotoxic properties of the mixed ligand gold(III) complexes were evaluated in vitro on human gastric SGC7901 and prostate PC3 cancer cells using MTT assay. The antiproliferative study of the gold(III) complexes on PC3 and SGC7901 cells indicate that complex 3 (containing 1S,2S-(+)-1,2-(DACH)) is the most effective antiproliferative agent. The IC50 data reveal that the in vitro cytotoxicity of complex 3 against SGC7901 cancer cells manifested similar and very pronounced cytotoxic effects with respect to cisplatin. Moreover, the electrochemical behavior, and the interaction of complex 3 with two well-known model proteins, namely, hen egg white lysozyme and bovine serum albumin is also reported.

Water-soluble Yb3+, Er3+ codoped NaYF4 nanoparticles induced SGC-7901 cell apoptosis through mitochondrial dysfunction and ROS-mediated ER stress.

BACKGROUND: Nanoparticles are potential luminescent probes; among them, upconversion nanoparticles (UCNP) are currently being developed as fluorescent probes for biomedical applications. However, the molecular mechanisms of UCNP in human gastric cell lines remain poorly understood. Here, we aimed to examine UCNP cytotoxicity to SGC-7901 cells and explore its underlying mechanisms. METHODS: The effects of 50-400 µg/mL UCNP on human gastric adenocarcinoma (SGC-7901) cells were investigated. Flow cytometry was used to evaluate reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), intracellular Ca2+ levels, and apoptosis. Activated caspase-3 and nine activities were measured; meanwhile, cytochrome C (Cyt C) in the cytosol and B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), protein kinase B (Akt), phosphorylated-Akt (p-Akt), 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), calpain-1, and calpain-2 protein levels were also detected. RESULTS: UCNP inhibited the viability of SGC-7901 cells in a concentration- and time-dependent manner and increased the proportion of cell apoptosis. Exposure to UCNP enhanced the ratio of Bax/Bcl-2, elevated the level of ROS, decreased ΔΨm, increased intracellular Ca2+ and Cyt C protein levels, decreased the levels of phosphorylated Akt, increased the activity of caspase-3 and caspase-9, and upregulated the protein expression of GRP-78, GRP-94, calpain-1 and calpain-2 in SGC-7901 cells. CONCLUSION: UCNP induced SGC-7901 cell apoptosis by promoting mitochondrial dysfunction and ROS-mediated endoplasmic reticulum (ER) stress, initiating the caspase-9/caspase-3 cascade.

Quinacrine Inhibits Cell Growth and Induces Apoptosis in Human Gastric Cancer Cell Line SGC-7901.

BACKGROUND: Quinacrine (QC), an antimalarial drug, has been shown to possess anticancer effect both in vitro (cancer cell lines) and in vivo (mouse models). In the cancer cells, QC can simultaneously suppress nuclear factor-κB and activate p53 signaling, which results in the induction of the apoptosis in these cells. However, the experimental results come from a few limited cancer cell lines, and the detailed mechanisms remain unknown. OBJECTIVE: This study investigated the tumor-killing effects of QC on gastric cancer cells as well as underlying molecular pathways. METHODS: SGC-7901 cells were treated with or without QC at different concentrations for 24 hours. The effect of QC on the inhibition of SGC-7901 cell proliferation was assessed by Cell Counting Kit-8 assay. Apoptosis was detected by examining nuclear morphology and quantifying phosphatidylserine externalization. Alterations in cellular morphology were analyzed by laser scanning confocal microscopy for fluorescent analysis. Cell cycle analysis was performed by propidium iodide (PI) staining and flow cytometry. The enzyme activity changes of caspase-3 were detected by colorimetry expression method. Western blot analysis was used to detect the changes in the protein level of Bax, Bc1-2, p53, and cytochrome c in cytosol of SGC-7901 cells. RESULTS: Our results showed that QC could significantly inhibit the growth of SGC-7901 cells in a dose-dependent manner, with the IC50 mean (SD) value of 16.18 (0.64) µM, compared with nontreated controls. QC treatment (15 µM) could also induce apoptosis in SGC-7901 cells (26.30% [5.31%], compared with control group of 3.37% [0.81%]; P < 0.01), and the increasing phosphatidylserine level and the accumulation of chromatin nucleation in QC-treated cells provided further evidence. In addition, cell cycle analysis with PI staining showed that a significant S enriches, increasing from 12.00% (1.24%) (control) to 20.94% (2.40%) (QC treatment) (P < 0.01). Furthermore, increased activities of caspase-3 (increasing from 0.108 [0.019] to 0.628 [0.068]; P < 0.01) were observed in SGC-7901 cells treated with 15 µM QC. Western blot analysis showed that QC treatment significantly increased the levels of proapoptotic proteins, including cytochrome c, Bax, and p53, and decreased the levels of antiapoptotic protein Bcl-2, thus shifting the ratio of Bax/Bcl-2 in favor of apoptosis. CONCLUSIONS: Our findings suggest that QC can significantly inhibit cell growth and induce apoptosis in SGC-7901 cells, which involves p53 upregulation and caspase-3 activation pathway.

Clinical significance and correlation of miR-200c and P-gp expression in gastric cancer and the effects on multidrug resistance.

Background: Poor prognosis is common in gastric cancer patients due to multidrug resistance (MDR)-induced recurrence and metastasis. In the present study, we investigated the expression of microRNA (miR)-200c in gastric cancer tissues and cell lines and its relationship with the expression of the drug resistant gene ABCB1, which encodes P-glycoprotein (P-gp). Methods: The basic characteristics of 102 patients with gastric cancer were reviewed. Real time-polymerase chain reaction (PCR), immunohistochemistry, and Western blot were employed to detect the expression levels of miR-200c and P-gp in gastric carcinoma tissues and cell lines. The correlation of miR-200c messenger RNA (mRNA) level with clinicopathological characteristics and P-gp protein expression were analyzed. SGC7901/vincristine (VCR) cells were transfected with miR-200c mimics or a specific small interfering RNA (siRNA) targeting the ABCB1 gene. The methyl thiazolyl tetrazolium (MTT) assay and flow cytometry were used to determine the role of miR-200c and ABCB1 on the viability and apoptosis of gastric carcinoma cell lines. Results: The level of miR-200c in carcinoma tissues was significantly lower than that in adjacent tissues, and the expression level of P-gp in carcinoma tissues was obviously higher than that in adjacent tissues (P<0.01, P=0.029). The expression levels of miR-200c and P-gp were associated with the malignant characteristics of gastric cancer, and patients with high expression of miR-200c or negative expression of P-gp had a better prognosis (P=0.006, P=0.022). MiR-200c negatively regulated the ABCB1 gene in gastric cancer cell lines. MiR-200c overexpression and ABCB1 down-regulation increased the sensitivity of SGC7901/VCR cells to VCR and reversed MDR by promoting cell apoptosis. Conclusions: The expression level of miR-200c decreases in gastric carcinoma tissues and drug-resistant gastric cancer SGC7901/VCR cells. Overexpression of miR-200c may enhance the sensitivity of SGC7901/VCR cells to VCR by regulating the expression of P-gp.

Celastrol Inhibits the Proliferation and Decreases Drug Resistance of Cisplatin- Resistant Gastric Cancer SGC7901/DDP Cells.

BACKGROUND: This study aimed to determine the effect and mechanism of Celastrol inhibiting the proliferation and decreasing the drug resistance of cisplatin-resistant gastric cancer cells. OBJECTIVE: The objective of this study was to explore the effect and mechanism of Celastrol on proliferation and drug resistance of human gastric cancer cisplatin-resistant cells SGC7901/DDP. METHODS: The thiazole blue (MTT) method was used to detect the sensitivity of human gastric cancer cisplatinresistant cells SGC7901/DPP to cisplatin and Celastrol to determine the Drug Resistance Index (DRI). According to the half Inhibitory Concentration (IC50) value, the action of the concentration of the following experimental drugs was set to reduce the cytotoxicity. Annexin V-FITC/PI double staining method was used to detect the apoptosis of SGC7901/DDP cells induced by Celastrol. Western Blot was used to examine the expression levels of P-glycoprotein (P-gp), Multidrug Resistance Associated Protein 1 (MRP1), Breast Cancer Resistance Associated Protein (Breast Cancer Resistance)-relative protein (BCRP), and mechanistic Target of Rapamycin (mTOR) pathway-related proteins. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of P-gp, MRP1, and BCRP. RESULTS: (1) Compared with the control group (we set the untreated group as the control group), the proliferation of the SGC7901/DPP cells was significantly inhibited after treating with 0.1-6.4µmol/L Celastrol in a time- and concentration-dependent manner (P<0.05). The Drug Resistance Index (DRI) of the SGC7901/DPP cells to DDP was 5.64. (2) Compared with the control group, Celastrol could significantly inhibit the proliferation and induce the apoptosis of the SGC7901/DPP cells (P<0.05). (3) The mRNA and protein expression levels of P-gp, MRP1, and BCRP in the SGC7901/DPP cells were significantly higher than those in the SGC7901 cells. However, after treating with Celastrol, the expression levels of P-gp, MRP1, and BCRP in the SGC7901/DPP cells were significantly reduced (P<0.05). (4) Compared with the control group, the Celastrol treatment also reduced the expression of the mTOR signaling pathway-related proteins, suggesting that the mTOR signaling pathway may be involved in the process of Celastrol inhibiting the proliferation of the SGC7901/DDP cells and reducing their drug resistance. (5) Significantly, the combination of Celastrol and DDP reduced the expression of P-gp, MRP1, and BCRP in the SGC7901/DPP cells. CONCLUSION: Celastrol can inhibit the proliferation of the SGC7901/DDP cells, induce their apoptosis, and reduce the expression of drug resistance genes, probably by inhibiting the expression of the proteins related to the mTOR signaling pathway.

miR-4486 enhances cisplatin sensitivity of gastric cancer cells by restraining the JAK3/STAT3 signalling pathway.

Along with the occurrence of cisplatin resistance, treatment on gastric cancer (GC) becomes difficult. Therefore, researches on new therapeutic methods to revert cisplatin resistance are becoming increasingly urgent. qRT-PCR was used to quantify the expression of miR-4486, JAK3 in SGC-7901 or SGC-7901/DDP cell lines. WB was utilized to analyze the expression of JAK3, STAT3 and p-STAT3 in SGC-7901/DDP cell lines. CCK-8 assay was used to determine the IC50 of cisplatin on both cell lines and cell viability of SGC-7901/DDP cell lines. The target relationship between miR-4486 and JAK3 was determined by luciferase assay. MiR-4486 expression on apoptosis of SGC-7901/DDP cell lines was determined by flow cytometry. qRT-PCR testified that miR-4486 decreased in SGC-7901/DDP cells, and the expression of miR-4486 mimic increased significantly compared with miR-4486 NC. By CCK-8 assay, the IC50 of cisplatin on both cell lines were 9 µg/mL and 81.3 µg/mL, and overexpression of miR-4486 decreased the viability of SGC-7901/DDP cells. Compared with DDP group, the expression of miR-4486 accelerated SGC-7901/DDP cells apoptosis. Dual-luciferase assay suggested that JAK3 was the target gene of miR-4486. qRT-PCR and WB proved that miR-4486/JAK3 axis inhibit the activation of JAK3/STAT3 pathway, and JAK3 overexpression can partly reverse this. As shown by CCK-8 and flow cytometry, miR-4486 overexpression decreased viability and stimulated apoptosis of SGC-7901/DDP cells. However, JAK3 overexpression can also partly revert this. miR-4486 overexpression could decrease viability and improve apoptosis of SGC-7901/DDP cells to revert its cisplatin-resistance, and the mechanism may be related to JAK3/STAT3 signalling pathway.