Parental Gastric Cancer Cells Research Articles

Yi-qi-hua-yu-jie-du decoction induces ferroptosis in cisplatin-resistant gastric cancer via the AKT/GSK3β/NRF2/GPX4 axis

BackgroundResistance to chemotherapy in gastric cancer (GC) is a ubiquitous challenge for its treatment. Yi-qi-hua-yu-jie-du decoction (YJD), an empirical formula in Traditional Chinese Medicine (TCM), demonstrated survival-prolonging functions in patients with GC. Previous research has shown that YJD could also inhibit drug resistance in GC. However, the precise mechanisms for how YJD accomplishes this remain incompletely explained. PurposeThe research aimed to identify differential metabolic characteristics in cisplatin-resistant GC and investigate whether YJD can target these differences to suppress GC drug resistance. MethodsMetabolomic analysis was conducted to identify metabolic disparities between cisplatin-resistant and parental GC cells, as well as metabolic modifications resulting from YJD intervention in cisplatin-resistant GC cells. The effect of YJD on ferroptosis stimulation was assessed by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA), iron ions, the reduced glutathione (GSH) to oxidised glutathione (GSSG) ratio, and alterations in mitochondrial morphology. Western blotting and quantitative real-time polymerase chain reaction (Q-PCR) were employed to verity the mechanisms of YJD-triggered ferroptosis through GPX4 and NRF2 overexpression models, alongside the AKT activator SC79. In vivo validation was conducted using nude mouse xenograft models. ResultsCisplatin-resistant GC exhibited altered GSH/GPX4 metabolism, and ferroptosis was a significantly enriched cell death pattern with YJD treatment in cisplatin-resistant GC cells. Ferroptosis biomarkers, including ROS, MDA, iron ions, the GSH/GSSG ratio, and mitochondrial morphology, were remarkably changed with the YJD intervention. Mechanistic experiments demonstrated that YJD inhibited the phosphorylation cascade activity of the AKT/GSK3β pathway, thereby reducing NRF2 expression. The level of GPX4, a crucial enzyme involved in glutathione metabolism, was attenuated, facilitating ferroptosis induction in cisplatin-resistant GC. ConclusionThe research reveals, for the first time, changes in GSH/GPX4 metabolism in cisplatin-resistant GC cells based on metabolomic analysis. YJD induced ferroptosis in cisplatin-resistant GC by inhibiting GPX4 through the AKT/GSK3β/NRF2 pathway, thus attenuating the cisplatin drug resistance in GC. Our findings identify metabolic changes in cisplatin-resistant GC and establish a theoretical framework for YJD on tackling drug resistance in GC through ferroptosis.

The CAGE-MiR-181b-5p-S1PR1 Axis Regulates Anticancer Drug Resistance and Autophagy in Gastric Cancer Cells.

Cancer-associated gene (CAGE), a cancer/testis antigen, has been known to promote anticancer drug resistance. Since the underlying mechanisms of CAGE-promoted anticancer drug resistance are poorly understood, we established Anticancer drug-resistant gastric cancer cells (AGSR) to better elucidate possible mechanisms. AGSR showed an increased expression level of CAGE and autophagic flux compared with anticancer drug-sensitive parental gastric cancer cells (AGS cells). AGSR cells showed higher invasion potential, growth rate, tumor spheroid formation, and angiogenic potential than AGS cells. CAGE exerted effects on the response to anticancer drugs and autophagic flux. CAGE was shown to bind to Beclin1, a mediator of autophagy. Overexpression of CAGE increased autophagic flux and invasion potential but inhibited the cleavage of PARP in response to anticancer drugs in CAGE CRISPR–Cas9 cell lines. TargetScan analysis was utilized to predict the binding of miR-302b-5p to the promoter sequences of CAGE, and the results show that miR-302b-5p directly regulated CAGE expression as illustrated by luciferase activity. MiR-302b-5p regulated autophagic flux and the response to anticancer drugs. CAGE was shown to bind the promoter sequences of miR-302b-5p. The culture medium of AGSR cells increased CAGE expression and autophagic flux in AGS cells. ImmunoEM showed CAGE was present in the exosomes of AGSR cells; exosomes of AGSR cells and human recombinant CAGE protein increased CAGE expression, autophagic flux, and resistance to anticancer drugs in AGS cells. MicroRNA array revealed miR-181b-5p as a potential negative regulator of CAGE. MiR-181b-5p inhibitor increased the expression of CAGE and autophagic flux in addition to preventing anticancer drugs from cleaving poly(ADP-ribose) polymerase (PARP) in AGS cells. TargetScan analysis predicted sphingosine 1-phosphate receptor 1 (SIPR1) as a potential target for miR-181b-5p. CAGE showed binding to the promoter sequences of S1PR1. The downregulation or inhibition of S1PR1 led to decreased autophagic flux but enhanced the sensitivity to anticancer drugs in AGSR cells. This study presents a novel role of the CAGE–miR-181b-5p–S1PR1 axis in anticancer drug resistance and autophagy.

TSPAN9 suppresses the chemosensitivity of gastric cancer to 5-fluorouracil by promoting autophagy

BackgroundThe issue of drug resistance in gastric cancer has attracted global attention. TSPAN9, a 4-transmembrane protein that plays an important role in tumor progression and signal transduction, has been found to be closely related to tumor invasion, metastasis, and autophagy.MethodsImmunoblotting was used to evaluate TSPAN9 expression in parental and drug-resistant gastric cancer cells. Functional assays, such as the CCK-8 assay, were used to detect the proliferation of gastric cancer cells and the response of TSPAN9 to 5-fluorouracil (5-FU). Western blotting was used to analyze the expression of constituents of the PI3K/AKT/mTOR-mediated autophagy pathway induced by TSPAN9. Coimmunoprecipitation was performed to assess the specific mechanism by which TSPAN9 affects the PI3K pathway.ResultsWe demonstrated that TSPAN9 is overexpressed in 5-FU-resistant cells compared to parental cells. 5-FU-mediated inhibition of cell proliferation can be significantly restored by increasing TSPAN9 expression, and inhibiting this expression in drug-resistant cells can restore the sensitivity of the cells to 5-FU. In addition, TSPAN9 also significantly promoted autophagy in gastric cancer cells in vitro. Further studies indicated that TSPAN9 downregulates the expression of PI3K and proteins associated with PI3K-mediated autophagy. In addition, TSPAN9 interacts with PI3K and inhibits its catalytic activity.ConclusionThe current study reveals the important role of TSPAN9 in drug resistance to 5-FU in gastric cancer. It also provides a new target to clinically address drug-resistant gastric cancer and will contribute to the treatment strategy of this disease.

DJ-1 overexpression confers the multidrug resistance phenotype to SGC7901 cells by upregulating P-gp and Bcl-2

Gastric cancer (GC) is one of the most malignant tumors with high incidence and mortality worldwide, and the multidrug resistance (MDR) often results in chemotherapy failure in GC. DJ-1 has been well indicated to be associated with drug resistance in multiple cancers. However, the role of DJ-1 in the MDR of gastric cancer cells and its possible mechanism remain to be elucidated. Therefore, the current study was investigated whether DJ-1 expression is differential in parental gastric cancer cell SGC7901 and vincristine (VCR)-induced gastric cancer MDR cell SGC7901/VCR, and whether DJ-1 plays a significant role in development of MDR in gastric cancer. The results showed that DJ-1 expression in SGC7901/VCR cells was significantly higher than its sensitive parental SGC7901 cells. Furthermore, DJ-1 overexpressed gastric cancer cell line SGC7901/LV-DJ-1 led to the increase of cell survival rate, the IC50 of chemotherapeutic drugs and number of cell clones as well as decrease of cell cycle G0/G1 phase ratio compared with its parental cells under the treatment of VCR, adriamycin (ADR), 5-Fluorouracil (5-FU) and cisplatin (DDP). However, the DJ-1 knockdown stable cell line SGC7901/VCR/shDJ-1 reversed the above mentioned series of MDR. Moreover, it was found that upregulation of DJ-1 protein expression promoted the pumping rate of GC cells to ADR and reduced the apoptotic index of GC cells treated with chemotherapeutic drugs by upregulating P-gp and Bcl-2. Similarly, knocking down DJ-1, P-gp or Bcl-2 displayed a converse effect. In conclusion, the current study demonstrated that DJ-1 overexpression confers the MDR phenotype to SGC7901 cells and this process is related to DJ-1 promoting active efflux of drugs and enhancing the anti-apoptotic ability of MDR GC cells by upregulating P-gp and Bcl-2.

RPPAs for Cell Subpopulation Analysis.

Understanding cellular heterogeneity is a challenge for cell biology, particularly in the fields of stem cell and cancer biology. We recently established a reverse phase protein array (RPPA)-modified method, colony lysate array (CoLA), which enables proteomic profiling of individual subpopulations (i.e., colonies) derived from various cell proliferative conditions. Here we describe two independent CoLA assays for a functional subpopulation, drug-tolerant persisters (DTPs), which are considered to mimic the origin of chemotherapeutic cancer relapse. In the first study, we analyzed individual DTPs derived from different cell types grown in the presence of different drugs. A hierarchical clustering analysis of DTPs using CoLA revealed two types of clonal populations, including those that expressed stem cell-associated proteins as well as epithelium-associated proteins. Subsequent principal component analyses demonstrated that the DTPs clustered on the basis of their proteomic profiles, which could change in response to drugs and doses. Another study was designed to identify signaling pathways that may be associated with drug resistance using a pair of 5-fluorouracil-tolerant and parental gastric cancer cell lines. Although a hierarchical clustering analysis did not show any specific clusters for the tolerant line, a drug dose-response analysis revealed that phosphorylation of PI3K and AKT increased and decreased, respectively, specifically in the tolerant cell line. These frameworks that readily profile small subpopulations isolated from an external stresses may be applicable to a wide variety of heterogeneous cellular samples.

Effect of long non-coding RNA EIF3J-AS1 on multi-drug resistance and autophagy in gastric cancer.

e15581 Background: Gastric cancer (GC) is a leading cause of cancer mortality worldwide, oxaliplatin and epirubicin based chemotherapy are one of the most important treatment options for GC patients. However, drug resistance, especially multi-drug resistance remains a major obstacle for successful chemotherapy. Recently, long non-coding RNAs (lncRNAs) have been widely identified to play emerging roles in diverse physiological and pathophysiological processes including drug resistance. Our previous bioinformatics analysis showed long non-coding RNA EIF3J-AS1 was a potential multi-drug resistance gene, but the underlying mechanism is still unknown. Methods: We generated oxaliplatin resistance cells (MGC803/OXA) and epirubicin resistance cells(MGC803/EPI) based on parental gastric cancer cells MGC803. Relative expression levels of EIF3J-AS1 were measured by qRT-PCR. Transmission electron microscopy was used to measure autophagosomes. Rapamycin was applied to inducing autophagy while chloroquine and 3-methyladenine were used to block autophagy. Protein level of autophagy related genes were examined by Western Blot. Coexpression genes of EIF3J-AS1 from TCGA RNA-seq datas were analyzed by cBiportal. RNA immunoprecipitation was used to analyze endogenous microRNAs and mRNAs. Results: EIF3J-AS1 was significantly upregulated in MGC803/OXA and MGC803/EPI cells compared with parental cells MGC803. EIF3J-AS1 inhibition increased chemosensitivity to both oxaliplatin and epirubicin. Moreover, EIF3J-AS1 silence lead to the decrease of autophagy. Autophagy related gene ATG14 was identified as a downstream target gene. EIF3J-AS1 promoted ATG14 expression by directly interacting with and increasing stability of ATG14 mRNA, On the other hand, EIF3J-AS1 competitively sponged miR-188-3p and promoted ATG14 expression in a ceRNA-dependent way. Conclusions: LncRNA EIF3J-AS1 is a crucial regulator of multi-drug resistance by inducing autophagy in gastric cancer. Targeting EIF3J-AS1/ATG14 axis might be a new paradigm for cancer therapeutics.

Tumorigenic hybrids between mesenchymal stem cells and gastric cancer cells enhanced cancer proliferation, migration and stemness.

BackgroundEmerging evidence indicates that inappropriate cell-cell fusion might contribute to cancer progression. Similarly, mesenchymal stem cells (MSCs) can also fuse with other cells spontaneously and capable of adopting the phenotype of other cells. The aim of our study was to investigate the role of MSCs participated cell fusion in the tumorigenesis of gastric cancer.MethodsWe fused human umbilical cord mesenchymal stem cells (hucMSCs) with gastric cancer cells in vitro by polyethylene glycol (PEG), the hybrid cells were sorted by flow cytometer. The growth and migration of hybrids were assessed by cell counting、cell colony formation and transwell assays. The proteins and genes related to epithelial- mesenchymal transition and stemness were tested by western blot、immunocytochemistry and real-time RT-PCR. The expression of CD44 and CD133 was examined by immunocytochemistry and flow cytometry. The xenograft assay was used to evaluation the tumorigenesis of the hybrids.ResultsThe obtained hybrids exhibited epithelial- mesenchymal transition (EMT) change with down-regulation of E-cadherin and up-regulation of Vimentin, N-cadherin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The hybrids also increased expression of stemness factors Oct4, Nanog, Sox2 and Lin28. The expression of CD44 and CD133 on hybrid cells was stronger than parental gastric cancer cells. Moreover, the migration and proliferation of heterotypic hybrids were enhanced. In addition, the heterotypic hybrids promoted the growth abilities of gastric xenograft tumor in vivo.ConclusionsTaken together, our results suggest that cell fusion between hucMSCs and gastric cancer cells could contribute to tumorigenic hybrids with EMT and stem cell-like properties, which may provide a flexible tool for investigating the roles of MSCs in gastric cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1780-1) contains supplementary material, which is available to authorized users.

MiR-223 promotes the cisplatin resistance of human gastric cancer cells via regulating cell cycle by targeting FBXW7.

BackgroundIncreasing evidence showed that miRNAs serve as modulators of human cancer, either as oncogene or tumor suppressors. Cisplatin resistance is the most common cause of chemotherapy failure in gastric cancer (GC). However, the roles of miRNAs in cisplatin resistance of GC remain largely unknown. The aim of the study was to identify a novel miRNA/gene pathway that regulates the sensitivity of GC cells to cisplatin.MethodsIn this study, we chose miR-223 by qRT-PCR analysis, the most significantly up-regulated miRNA in GC, to investigate its formation of DDP-resistant phenotype of GC cells and possible molecular mechanisms.ResultsWe found that miR-223 was most significantly up-regulated miRNA in DDP-resistant GC cells compared with parental GC cells. Besides, its expression was also significantly up-regulated in GC tissues. FBXW7 was identified as the direct and functional target gene of miR-223. Overexpression of FBXW7 could mimic the effect of miR-223 down-regulation and silencing of FBXW7 could partially reverse the effect of miR-223 down-regulation on DDP resistance of DDP-resistant GC cells. Besides, miR-223 and FBXW7 could affect the G1/S transition of cell cycle by altering some certain cell cycle regulators. Furthermore, miR-223 was found to be significantly up-regulated in H. pylori infected tissues and cells, suggesting that H. pylori infection may lead to GC development and DDP resistance.ConclusionsOur findings revealed the roles of miR-223/FBXW7 signaling in the DDP resistance of GC cells and targeting it will be a potential strategic approach for reversing the DDP resistance in human GC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0145-6) contains supplementary material, which is available to authorized users.

Abstract 1444: Sensitivity to trastuzumab for gastric cancer is regulated by miR-223/FBXW7 pathway

Abstract Background: A recent large-scale phase III study (the ToGA trial) demonstrated the significant efficacy of trastuzumab in addition to chemotherapy in patients with HER2-positive gastric cancer (GC). Although trastuzumab has become a key drug in recent cancer treatment, resistance to trastuzumab for breast cancer is a major problem in clinical practice. However, it is unclear whether similar mechanisms of trastuzumab resistance act in GC. F-box and WD repeat domain-containing 7 (FBXW7) is the substrate recognition components of an evolutionarily conserved SCF (complex of SKP1, CUL1, and F-box protein)-type ubiquitin ligase complexe, which has been well characterized and shown to have important roles in regulating the stability of multiple oncoprotein substrates, including cyclin E, c-Myc, Notch, c-Jun, mTOR, and MCL1. Therefore, FBXW7 is recognized to be one of the major causes of drug resistance. The aim of the current study is to clarify the novel microRNA (miR)-FBXW7 pathway regulating resistance to trastuzumab. Method: Using 5 GC cell lines, we examined the following items in vitro. 1. We examined the status of FBXW7 expression by quantitative real-time reverse transcription-PCR (qRT-PCR). 2. We established the trastuzumab resistance GC cell line with continuous trastuzumab administration. Parent GC cell line and resistance GC cell line are subjected to miR PCR array. 3. We focused on miR223-FBXW7 pathway. We examined the status of HER2, miR223, FBXW7, and its target gene MCL1 expression by up or down regulation of miR-223 in GC cell lines. 4. We investigated whether the miR-223-FBXW7 pathway can affect the resistance of trastuzumab. Result: FBXW7 and miR-223 expression showed inverse correlation. Moreover, miR-223 expression was increased and FBXW7 expression was decreased whereas MCL1 expression, downstream targets of FBXW7, was increased while no significant change was observed both in HER2 and p-HER2 in GC trastuzumab resistance cells. Overexpression of miR-223 decreased FBXW7 expression persisting HER2 and p-HER2 expression and can induce the resistance of trastuzumab. The suppression of miR-223 increased FBXW7 expression persisting HER2 and p-HER2 expression and can simultaneously restore the sensitivity of trastuzumab.Conclusion: Our current study revealed for the first time that regulated the sensitivity of HER2-positive GC cell line to trastuzumab. These findings suggest that this pathway may be crucial to the mechanism of resistance to trastuzumab in GC, which may lead to the development of individualized treatment in clinical practice. Citation Format: Kojiro Eto, Masaaki Iwatsuki, Takatsugu Ishimoto, Hideo Baba. Sensitivity to trastuzumab for gastric cancer is regulated by miR-223/FBXW7 pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1444. doi:10.1158/1538-7445.AM2014-1444

Genomic analysis of drug resistant gastric cancer cell lines by combining mRNA and microRNA expression profiling

Understanding the mechanism underlying multidrug resistance and identifying effective targets that can overcome it is of critical importance. In this study, mRNA and miRNA expression profiling of the drug resistant sublines, SGC7901/VCR and SGC7901/ADR, and their parental gastric cancer cell line SGC7901 were performed. A significant number of genes and a limited subset of miRNAs were commonly dysregulated, which were further validated using qRT-PCR. GO and KEGG pathway analyses of the commonly dysregulated genes indicated that the MAPK signalling pathway may be involved in multidrug resistance, which was further validated using immunoblotting and MTT assay. Finally a primary multidrug resistance network in gastric cancer, consisting of the commonly dysregulated genes and miRNAs, was established and functional miRNA–mRNA pairs were identified. The commonly dysregulated genes and miRNAs identified in this study may represent good therapeutic targets and further study of these targets may increase our understanding of the mechanisms underlying multidrug resistance.

VEGF‐C mediates RhoGDI2‐induced gastric cancer cell metastasis and cisplatin resistance

Rho GDP dissociation inhibitor 2 (RhoGDI2) expression is correlated with tumor growth, metastasis and chemoresistance in gastric cancer. However, the mechanisms by which RhoGDI2 promotes tumor cell survival and metastasis remain unclear. In this study, we clearly demonstrate that RhoGDI2 upregulates VEGF-C expression and RhoGDI2 expression is positively correlated with VEGF-C expression in human gastric tumor tissues as well as parental gastric cancer cell lines. VEGF-C depletion suppressed RhoGDI2-induced gastric cancer metastasis and sensitized RhoGDI2-overexpressing cells to cisplatin-induced apoptosis in vitro and in vivo. Secreted VEGF-C enhanced gastric cancer cell invasion and conferred cisplatin resistance to RhoGDI2-overexpressing cells. We also show that RhoGDI2 positively regulates Rac1 activity in gastric cancer cells. Inhibition of Rac1 expression suppressed RhoGDI2-induced VEGF-C expression, and this inhibition was associated with decreased invasiveness and increased sensitivity to cisplatin in RhoGDI2-overexpressing cells. Our results indicate that RhoGDI2 might be a potential therapeutic target for simultaneously reducing metastasis risk and enhancing chemotherapy efficacy in gastric cancer.

The mechanism of COX-2 regulating HERG channel in gastric cancer cells.

To elucidate the signal transduction pathway, by which cyclooxygenase-2 (COX-2) regulates human erg-related gene (HERG) current in gastric cancer cells. The HERG mRNA, protein and current in gastric cancer cells transfected with or without COX-2 antisense vector were measured by RT-PCR, Western blot and patch-clamp, respectively. Cyclic adenosine monophosphate (cAMP) concentration in gastric cancer cells transfected with or without COX-2 antisense vector was measured by ELISA. Transfection with COX-2 antisense vector did not alter the expression of HERG mRNA and protein, but it diminished the amplitude of HERG current in gastric cancer cells (p < 0.05). The cAMP concentration in gastric cancer cells transfected with COX-2 antisense vector was lower than that in parental gastric cancer cells (p < 0.05). COX-2 inhibitor and PGE2 had influence on the HERG current in gastric cancer cells. COX-2 inhibitor reduced the amplitude of HERG current in gastric cancer cells and PGE2 enhanced the amplitude. However, in gastric cancer cells transfected with HERG mutant deleting cAMP-binding domain, both COX-2 inhibitor and PGE2 did not show significant effects on HERG current. cAMP agonist enhanced the amplitude of HERG current and cAMP antagonist reduced the amplitude in gastric cancer cells. Both agonist and antagonist of cAMP had no significant effect on HERG current in gastric cancer cells transfected with HERG mutant deleting cAMP binding domain. PKA inhibitor did not influence the HERG current whether in parental gastric cancer cells or in gastric cancer cells transfected with HERG mutant.

Enhancement of 5-fluorouracil-induced cytotoxicity by leucovorin in 5-fluorouracil-resistant gastric cancer cells with upregulated expression of thymidylate synthase

BackgroundElucidation of the mechanisms by which gastric cancer cells acquire resistance to 5-fluorouracil (5FU) may provide important clues to the development of effective chemotherapy for 5FU-resistant gastric cancerMethodsFour 5FU-resistant cell lines (MKN45/5FU, MKN74/5FU, NCI-N87/5FU, and KATOIII/5FU) were established by continuous exposure of the cells to progressively increasing concentrations of 5FU for about 1 year. Then, mRNA expression levels of four genes associated with 5FU metabolism, i.e., thymidylate synthase (TS), dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase, were quantitatively evaluated by real-time reverse transcriptase-polymerase chain reaction. In addition, TS protein expression was measured by Western blot analysis.ResultsAs compared with the parent cell lines, the 5FU-resistant cell lines showed 3.8- to 11.6-fold higher resistance to 5FU, as well as 1.9- to 3.5-fold higher TS mRNA expression and 1.6- to 7.1-fold higher TS protein expression. In contrast, the expressions of other genes did not differ significantly among the cell lines. The cytotoxicity of 5FU was enhanced 2.3- to 2.8 fold by leucovorin (LV) against three of the four 5FU-resistant cell lines.ConclusionsCollectively, LV enhanced the cytotoxicity of 5FU not only against the parent gastric cancer cell lines, but also against the 5FU-resistant cell lines, even those with elevated TS expression levels. These results suggest that clinical studies of a combination of 5FU and LV are warranted in patients who have recurrent gastric cancer after 5FU-based therapy.

642 VEGF-C Mediates RhoGDI2-induced Gastric Cancer Cell Metastasis and Cisplatin Resistance

Rho GDP dissociation inhibitor 2 (RhoGDI2) expression is correlated with tumor growth, metastasis and chemoresistance in gastric cancer. However, the mechanisms by which RhoGDI2 promotes tumor cell survival and metastasis remain unclear. In this study, we clearly demonstrate that RhoGDI2 upregulates VEGF-C expression and RhoGDI2 expression is positively correlated with VEGF-C expression in human gastric tumor tissues as well as parental gastric cancer cell lines. VEGF-C depletion suppressed RhoGDI2-induced gastric cancer metastasis and sensitized RhoGDI2-overexpressing cells to cisplatin-induced apoptosis in vitro and in vivo. Secreted VEGF-C enhanced gastric cancer cell invasion and conferred cisplatin resistance to RhoGDI2-overexpressing cells. We also show that RhoGDI2 positively regulates Rac1 activity in gastric cancer cells. Inhibition of Rac1 expression suppressed RhoGDI2-induced VEGF-C expression, and this inhibition was associated with decreased invasiveness and increased sensitivity to cisplatin in RhoGDI2-overexpressing cells. Our results indicate that RhoGDI2 might be a potential therapeutic target for simultaneously reducing metastasis risk and enhancing chemotherapy efficacy in gastric cancer.

Abstract 780: An EGFR inhibitor enhances the efficacy of SN38, an active metabolite of irinotecan, in SN38-refractory gastric carcinoma cells

Abstract Background: Acquired drug resistance to irinotecan is one of the significant obstacles in the treatment of advanced gastric cancer. This study was performed to clarify the effect of epidermal growth factor receptor (EGFR) inhibitors in combination with SN38, an active metabolite of irinotecan, on the proliferation of irinotecan-refractory gastric cancer. Methods: Two irinotecan-resistant gastric cancer cell lines, OCUM-2M/SN38 and OCUM-8/SN38, were respectively established by stepwise exposure to SN38 from the parent gastric cancer cell lines OCUM-2M and OCUM-8. The combination effects of two EGFR inhibitors, gefitinib and lapatinib, with SN38 on proliferation, apoptosis, and cell-cycle on gastric cancer cells were examined. Results: Gefitinib or lapatinib showed synergistic anti-tumor effects against OCUM-2M/SN38 and OCUM-8/SN38 cells when used in combination with SN38, but not against OCUM-2M or OCUM-8 cells. SN38 increased the expression of EGFR and HER2 in OCUM-2M/SN38 and OCUM-8/SN38 cells. The combination of an EGFR inhibitor and SN38 significantly increased the levels of apoptosis-related molecules, caspase-6, p53, and DAPK-2, and resulted in the induction of apoptosis of irinotecan-resistant cells. The EGFR inhibitors increased the S-phase and decreased the UGT1A1 and ABCG expression in irinotecan-resistant cells. The SN38 plus Lapatinib group more effectively suppressed in vivo tumor growth by OCUM-2M/SN38 cells than either alone group.Conclusion: The combination treatment with an EGFR inhibitor and irinotecan might produce synergistic anti-tumor effects for irinotecan-refractory gastric cancer cells. The regulation of SN38 metabolism-related genes and cell cycle by EGFR inhibitors might be responsible for the synergism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 780. doi:1538-7445.AM2012-780

PA28β regulates cell invasion of gastric cancer via modulating the expression of chloride intracellular channel 1

PA28β is a subunit of proteasome activator PA28. Previous study suggests that PA28β is involved in the invasiveness and metastasis of gastric adenocarcinoma (GA), however, the mechanism is not fully understood. In the present study, we showed that invasive abilities of gastric cancer cells were enhanced when PA28β being down-regulated, and were inhibited when PA28β being overexpressed. To explore the possible mechanism of PA28β associated elevated invasiveness, the protein profiles of PA28β knock down and parental negative control gastric cancer cells were compared using proteomics approach. The results revealed that there were 43 proteins were differentially expressed, among them, chloride intracellular channel 1 (CLIC1) was significantly up-regulated and selected for further functional study. Down-regulation of CLIC1 by RNA interference was able to markedly inhibit cell invasion of PA28β knock down gastric carcinoma cells. In addition, an inverse correlation between PA28β and CLIC1 expressions was also verified in GA tissue samples, suggesting that knockdown of PA28β could enhance tumor invasion and metastasis, at least in part, through up-regulation of CLIC1. Our results provide novel insight into the mechanisms of PA28β related invasiveness and metastasis of GA, and suggest new alternative approaches for GA treatment.

An EGFR inhibitor enhances the efficacy of SN38, an active metabolite of irinotecan, in SN38-refractory gastric carcinoma cells

Background:Acquired drug resistance to irinotecan is one of the significant obstacles in the treatment of advanced gastric cancer. This study was performed to clarify the effect of epidermal growth factor receptor (EGFR) inhibitors in combination with SN38, an active metabolite of irinotecan, on the proliferation of irinotecan-refractory gastric cancer.Methods:Two irinotecan-resistant gastric cancer cell lines, OCUM-2M/SN38 and OCUM-8/SN38 were, respectively, established by stepwise exposure to SN38 from the parent gastric cancer cell lines OCUM-2M and OCUM-8. The combination effects of two EGFR inhibitors, gefitinib and lapatinib, with SN38 on proliferation, apoptosis, and cell cycle on gastric cancer cells were examined.Results:Gefitinib or lapatinib showed synergistic anti-tumour effects against OCUM-2M/SN38 and OCUM-8/SN38 cells when used in combination with SN38, but not against OCUM-2M or OCUM-8 cells. SN38 increased the expression of EGFR and HER2 in OCUM-2M/SN38 and OCUM-8/SN38 cells. The combination of an EGFR inhibitor and SN38 significantly increased the levels of apoptosis-related molecules, caspase-6, p53, and DAPK-2, and resulted in the induction of apoptosis of irinotecan-resistant cells. The EGFR inhibitors increased the S-phase and decreased the UGT1A1 and ABCG expression in irinotecan-resistant cells. The SN38 plus Lapatinib group more effectively suppressed in vivo tumour growth by OCUM-2M/SN38 cells than either alone group.Conclusion:The combination treatment with an EGFR inhibitor and irinotecan might produce synergistic anti-tumour effects for irinotecan-refractory gastric cancer cells. The regulation of SN38 metabolism-related genes and cell cycle by EGFR inhibitors might be responsible for the synergism.

A FGFR2 inhibitor, Ki23057, enhances the chemosensitivity of drug-resistant gastric cancer cells

AimThe aim of this study was to clarify the ability of a FGFR2 inhibitor, Ki23057, to enhance the chemosensitivity of drug-resistant gastric cancer cell lines when used in combination with chemotherapeutic drugs. Materials and methodsFive cancer cell lines resistant to irinotecan (SN38), paclitaxel (PTX), etoposide (VP16), oxaliplatin (OXA), and gemcitabine (GEM) were respectively established from a parent gastric cancer cell line, OCUM-2M, and were named OCUM-2M/SN38, OCUM-2M/PTX, OCUM-2M/VP16, OCUM-2M/OXA, and OCUM-2M/GEM. The effects of the combination of Ki23057 with anticancer drugs on proliferation, apoptosis, and mRNA expression were examined. ResultsKi23057 significantly decreased the IC50 values of OCUM-2M/SN38, OCUM-2M/PTX, and OCUM-2M/VP16, but not those of OCUM-2M/OXA and OCUM-2M/GEM. Ki23057 significantly enhanced the apoptosis rates induced by chemotherapeutic drugs in both the drug-resistant cell lines and the parental cell line. Ki23057 decreased the ERCC1 expression level in OCUM-2M/SN38, OCUM-2M/PTX, and OCUM-2M/VP16. Ki23057 increased the p53 expression level in OCUM-2M/SN38 and OCUM-2M/PTX, but not in OCUM-2M/VP16. ConclusionThe FGFR2 inhibitor Ki23057 might be therapeutically promising for treating drug-resistant gastric cancer cells, especially when used in combination with SN38, PTX, or VP16. The apoptosis process might be the main mechanism underlying the synergistic effect of these combinations. The ERCC1 and p53 genes may play an integral role in the synergism between Ki23057 and chemotherapeutic agents in drug-resistant cell lines.

Lentiviral vector-mediated upregulation of let-7a inhibits gastric carcinoma cell growth in vitro and in vivo

Objective. Let-7a, a molecule that is reduced in various cancers, has been associated with cell growth and proliferation. Upregulation of let-7a may inhibit tumor cell growth. To verify this hypothesis, let-7a gene overexpression was studied in gastric cancer cells in vitro and in vivo. Material and methods. A lentiviral system harboring both enhanced green fluorescent protein reporter gene and the let-7a short hairpin RNA expression cassette was firstly constructed. Then the stable let-7a gene overexpression SGC-7901 cells were established and real-time RT-PCR analysis was used to evaluate the expression of the let-7a gene. Their growth-inhibiting, cell cycle arrest characteristics were identified. Results. In SGC-7901/let-7a cells, let-7a expression was significantly increased. Their proliferation was obviously retarded and the cell cycle changed with increased G0/G1 arrest and decreased S phase. In animal experiments, SGC-7901/let-7a cell xenografts demonstrated growth suppression compared to parental or control gastric cancer cell xenografts. Conclusion. The results demonstrated that lentiviral vector was capable of upregulating let-7a, resulting in impressive anticancer effects. It offers a powerful new strategy for the development of novel therapeutic interventions to treat human gastric carcinomas.

Anti-tumor effects of CIK combined with oxaliplatin in human oxaliplatin-resistant gastric cancer cells in vivo and in vitro

BackgroundDrug resistance remains a great challenge in the treatment of gastric cancer. The goal of this study was to explore the anti-tumor effects and mechanism of cytokine-induced killer (CIK) cell combined with oxaliplatin (L-OHP) in human oxaliplatin-resistant gastric cancer cells.MethodsAfter producing oxaliplatin-resistant gastric cancer cells, cell morphology, growth and doubling time were observed, followed by detection of cell cycle distribution and apoptosis, drug sensitivity (e.g., L-OHP) and expression of P-gp and livin. MTT assay, in vivo pharmacodynamics and pathomorphology experiments were used to detect killing activities of CIK combined with L-OHP.ResultsCompared with parental gastric cancer cells, oxaliplatin-resistant gastric cancer cells in S phase were reduced and cell apoptosis rate was increased (P < 0.05), the inhibition rate of 10 chemotherapeutics on oxaliplatin-resistant gastric cancer cells was significantly lower and the expression of P-gp was significantly higher (P < 0.05). However, there was no significant difference in livin expression between parental gastric cancer cells and oxaliplatin-resistant gastric cancer cells (P > 0.05). The in vitro killing activity of CIK combined with L-OHP on parental cells and oxaliplatin-resistant cells were significantly enhanced compared with L-OHP or CIK alone. And it showed greater synergetic effects against oxaliplatin-resistant cells compared with parental cells (P < 0.05). In addition, survival rate, abdominal circumference and pathomorphology results revealed stronger in vivo anti-tumor effects when the two therapies were combined.ConclusionsThe mechanism of oxaliplatin-resistant cell secondary multidrug resistance was correlated with the variation of cell cycle distribution, extension of doubling time and upregulation of P-gp expression. The synergistic effect of CIK in combination with L-OHP on killing activity against oxaliplatin-resistant cells was shown in vivo and in vitro.