Resistance Of Gastric Cancer Cells Research Articles

Abstract 3600: Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation

Abstract The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C14-labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells. Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines. Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2015-3600

Abstract 2539: Targeting therapy-resistant gastric and esophageal cancers with mTOR ATP site inhibitors

Abstract Introduction: Despite a declining incidence, gastroesophagealcancer remains a leading cause of cancer death in the Western world. At time of diagnosis, most patients have advanced disease, for which treatment goals are palliative and options are limited. Available cytotoxic chemotherapy have not been successful in durable response or improving the survival… Developing rationale and novel therapy in gastroesophageal cancers is clearly an area of unmet need. Tumor recurrence is the persistence of a rare sub-population of cells with tumorigenic potential and stem cell markers that is intrinsically resistant to conventional therapy. Tumor-initiating or cancer stem cells have been described in many cancers, both hematologic and solid malignancies. Improving survival in patients with gastroesophageal cancer will require development of therapeutic agents targeting tumor initiating while, at the same time, sparing normal stem cells. Achieving this important goal will require increased knowledge of the signals required for maintenance and expansion of cancer vs. normal stem cells. Recent investigation by our group has demonstrated that ribosome biogenesis pathways have a major role to play in cancer and tumor initiation. mTOR plays a critical role in modulating the role of ribosomes in cancer, specifically its translational arm which can selectively regulate the translational complex in these cells and affecting their ability to proliferate, invade and metastasisze. Methods: Our goal is to study the role of mTOR pathway in treatment resistant gastric/GEJ cancer and target the same using ATP-site inhibitors of mTOR which target both the proliferation as well as the translational arm of mTOR. Results: Our results demonstrates that by targeting mTOR using ATP site inhibitors in therapy resistant gastric (KATOIII and NCI-N87) and esophageal cancer cell lines (KYSE30 and KYSE270) significantly (p<0.05) reduces proliferation. Combination of docetaxel and mTOR ATP site inhibitor led to dramatic reduction in proliferation in a dose dependent manner. Addition assays determined that reduced proliferation corresponds to an increase in apoptosis, which suggests that this cell killing may not cause major toxicity. We are currently examining the effects of these combinations in vivo. Conclusions: The combination of standard of care and mTOR ATP side inhibitor is potentially effective in reducing cell proliferation using apoptosis as the means of cell death. Our preliminary findings suggest that targeting the translational as well as the cell size arm of the mTOR signaling pathway may be a rationale and novel strategy to treat gastroesophageal cancer. Citation Format: Bhuvanesh Dave, Alexandria Phan. Targeting therapy-resistant gastric and esophageal cancers with mTOR ATP site inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2539. doi:10.1158/1538-7445.AM2015-2539

GTSE1 expression represses apoptotic signaling and confers cisplatin resistance in gastric cancer cells

BackgroundPlatinum based therapy is commonly used in the treatment of advanced gastric cancer. However, resistance to chemotherapy is a major challenge that causes marked variation in individual response rate and survival rate. In this study, we aimed to identify the expression of GTSE1 and its correlation with cisplatin resistance in gastric cancer cells.MethodsMethylation profiling was carried out in tissue samples from gastric cancer patients before undergoing neoadjuvent therapy using docetaxel, cisplatin and 5FU (DCX) and in gastric cancer cell lines. The correlation between GTSE1 expression and methylation in gastric cancer cells was determined by RT-PCR and MSP respectively. GTSE1 expression was knocked-down using shRNA’s and its effects on cisplatin cytotoxicity and cell survival were detected by MTS, proliferation and clonogenic survival assays. Additionally, the effect of GTSE1 knock down in drug induced apoptosis was determined by western blotting and apoptosis assays.ResultsGTSE1 exhibited a differential methylation index in gastric cancer patients and in cell lines that correlated with DCX treatment response and cisplatin sensitivity, respectively. In-vitro, GTSE1 expression showed a direct correlation with hypomethylation. Interestingly, Cisplatin treatment induced a dose dependent up regulation as well as nuclear translocation of GTSE1 expression in gastric cancer cells. Knock down of GTSE1 enhanced cisplatin cytotoxity and led to a significant reduction in cell proliferation and clonogenic survival. Also, loss of GTSE1 expression caused a significant increase in P53 mediated apoptosis in cisplatin treated cells.ConclusionOur study identifies GTSE1 as a biomarker for cisplatin resistance in gastric cancer cells. This study also suggests the repressive role of GTSE1 in cisplatin induced apoptosis and signifies its potential utility as a therapeutic target for better clinical management of gastric cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1550-0) contains supplementary material, which is available to authorized users.

Biased random walk model for the prioritization of drug resistance associated proteins.

Multi-drug resistance is the main cause of treatment failure in cancer patients. How to identify molecules underlying drug resistance from multi-omics data remains a great challenge. Here, we introduce a data biased strategy, ProteinRank, to prioritize drug-resistance associated proteins in cancer cells. First, we identified differentially expressed proteins in Adriamycin and Vincristine resistant gastric cancer cells compared to their parental cells using iTRAQ combined with LC-MS/MS experiments, and then mapped them to human protein-protein interaction network; second, we applied ProteinRank to analyze the whole network and rank proteins similar to known drug resistance related proteins. Cross validations demonstrated a better performance of ProteinRank compared to the method without usage of MS data. Further validations confirmed the altered expressions or activities of several top ranked proteins. Functional study showed PIM3 or CAV1 silencing was sufficient to reverse the drug resistance phenotype. These results indicated ProteinRank could prioritize key proteins related to drug resistance in gastric cancer and provided important clues for cancer research.

Molecular mechanism of cisplatin to enhance the ability of TRAIL in reversing multidrug resistance in gastric cancer cells

To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells. MTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis. After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations. The activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

DBC1 promotes anoikis resistance of gastric cancer cells by regulating NF-κB activity.

Deleted in breast cancer 1 (DBC1) has been known to be overexpressed and serves as a poor prognostic indicator of several human cancers. In this study, we examined DBC1 expression in a total of 142 gastric cancer tissues by immunohistochemistry and revealed that DBC1 was overexpressed in gastric cancer and significantly associated with TNM stage and lymph node metastasis. The in vitro experiments showed that DBC1 expression correlated with the ability of anoikis resistance in gastric cancer cells, which has been defined as critical to metastasis. Furthermore, the results showed that the IKK-β/NF-κB signaling pathway was involved in the regulation of anoikis resistance by DBC1 in gastric cancer cells. Taken together, the results indicated that DBC1 promotes anoikis resistance in gastric cancer cells by regulating NF-κB activity and may thus be a new therapeutic target for preventing potential metastasis.

Cetuximab-induced insulin-like growth factor receptor I activation mediates cetuximab resistance in gastric cancer cells

Epidermal growth factor receptor (EGFR) and insulin‑like growth factor receptor‑I (IGF‑IR) are frequently overexpressed in gastric cancercells. However, thesecells are resistant to the anti‑EGFR monoclonal antibody cetuximab. The aim of the present study was to determine whether cetuximab resistance in gastric cancercells resulted from activation of the IGF‑IR signaling pathway by cetuximab. The results demonstrated that EGFR phosphorylation was markedly inhibited in gastric cancer cell lines (SGC7901 and MGC803) which possessed functional K‑ras and BRAF following treatment with cetuximab. However, cetuximab treatment did not diminish cell viability; by contrast, IGF‑IR activation was observed. Knockdown of IGF‑IR or the use of an IGF‑IR inhibitor were found to increase the sensitivity of gastric cancercells to cetuximab. Furthermore, cetuximab induced phosphorylation of the non‑receptor tyrosine kinase c‑steroid receptor co‑activator (Src). Treatment of gastric cancercells with a Src inhibitor was shown to significantly reduce cetuximab‑induced phosphorylation of IGF‑IR as well as Src, which resulted in enhanced sensitivity to cetuximab treatment. In conclusion, the results of the present study demonstrated that cetuximab‑induced IGF‑IR activation was involved in cetuximab resistance in gastric cancercells and that Src was an important mediator for IGF‑IR activation.

TXNL1 induces apoptosis in cisplatin resistant human gastric cancer cell lines.

Cisplatin is one of the most commonly used drugs in the treatment of gastric cancer. However, drug resistance is a major obstacle for effective treatment and originates in multiple mechanisms such as enhanced DNA repair and anti-apoptosis. Our previous results demonstrated that XRCC1 was a key regulator of cisplatin induced DNA damage and apoptosis. TXNL1, a member of the thioredoxin family, negatively regulated the expression of XRCC1 via the ubiquitin-proteasome pathway. Here, we investigated the role of TXNL1 in the apoptosis induced by cisplatin. Our data showed that the expression of TXNL1 in the cisplatin resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP cells was significantly lower compared with the cisplatin sensitive cell lines BGC823 and SGC7901. Inhibition of the expression of TXNL1 in BGC823 and SGC7901 cells led to increased resistance to cisplatin induced apoptosis and cell death detected by Tunel and clonogenic assay, respectively. In contrast, over expression of TXNL1 in BGC823/DDP and SGC7901/DDP cells lead to higher cisplatin induced apoptosis and cell death. Moreover, our results demonstrated that the mechanism of TXNL1 regulating cisplatin-induced apoptosis was closely associated with Bcl-2 mediated mitochondria apoptosis pathway. In conclusion, these findings suggest that TXNL1 was a feasible modulator and potential chemotherapeutic target for the cisplatin resistant phenotype of human gastric cancer cells.

Effect of Vav 3 gene inhibition on inhibitor of apoptosis proteins and multidrug resistance in gastric cancer cells

Objective To explore the role of Vav3 gene in multidrug resistance(MDR)of gastric cancer and the related mechanism.Methods QRT-PCR was used to examine the expressions of Vav3 gene in gastric cancer tissues,tumor-adjacent tissues,human gastric cancer cell line SGC7901,and gastric epithelial cell line GES-1.Then Vav3-siRNA was synthesized and tansfected into SGC7901 cells. MTT assay was then used to determine the inhibition rates of tumor cells exposed to chemotherapeutic agents(5-FU,L-OHP)before and after Vav3-siRNA transfection.Real-time RT-PCR and Western blotting analysis were used to observe the expressions of inhibitor of apoptosis proteins(IAPs):xIAP,Survivin,and Livin;meanwhile,the expression and activity of Caspase-3and Caspase-8 were also determined.Results Vav3 was over-expressed in gastric cancer tissues and gastric cell line compared with those in tumor-adjacent tissues and gastric epithelial cell line GES-1(P0.05).Expression of Vav3 was significantly inhibited by Vav3-siRNA(P0.01).Inhibition rates of tumor cells exposed to 5-FU and L-OHP were significantly increased 48 hafter Vav3-siRNA tansfection(P0.05).The expressions of xIAP and Survivin were significantly decreased in cancer cells after Vav3-siRNA tansfection(both P0.05),and no notable change was found for Livin expression;also the expression and activity of Caspase-3and Caspase-8protein were significantly increased after Vav3-siRNA tansfection in SGC7901cells(all P0.05).Conclusion Vav3 can participate in MDR of gastric cancer by regulating apoptotic pathways,and inhibition of Vav3 can help reverse MDR of gastric cancer cells by regulating some IAPs.

MnSOD overexpression confers cisplatin resistance in lung adenocarcinoma via the NF-κB/Snail/Bcl-2 pathway

Manganese superoxide dismutase (MnSOD) has been shown to be associated with doxorubicin resistance in gastric cancer cells, but the underlying mechanism of MnSOD in drug resistance remains unclear. A recent study indicated that NF-κB activation by MnSOD promoted tumor malignancy in lung adenocarcinoma. Therefore, we hypothesized that MnSOD-mediated NF-κB activation might confer cisplatin resistance in lung adenocarcinoma via the NF-κB/Bcl-2/Snail pathway. Here, the inhibition concentration of cisplatin with 50% cell viability (IC50) was positively correlated with MnSOD expression and its activity in a panel of lung adenocarcinoma cells. The IC50 value was markedly increased and decreased by MnSOD overexpression and knockdown, respectively, in lung cancer cells. Mechanistically, an increase in Bcl-2 by MnSOD-mediated NF-κB activation confers greater cisplatin resistance than cIAP2, Bcl-xL, Mcl-1, and Snail. MnSOD-mediated cisplatin resistance can be overcome by a Bcl-2 antagonist (ABT-199) or IKKβ inhibitor (curcumin) in cells and xenograft tumors. MnSOD expression was positively correlated with nuclear p65 protein and Bcl-2 mRNA expression in tumors from patients with lung adenocarcinomas. A retrospective study indicated that it was more common for MnSOD-positive, nuclear p65-positive, or high Bcl-2 mRNA tumors to have an unfavorable response to cisplatin-based chemotherapy than their counterparts. Therefore, we suggest that ABT-199 or curcumin may be potentially useful to improve tumor regression and chemotherapeutic response in patients with MnSOD/Bcl-2-positive tumors.

Involvement of miR-143 in cisplatin resistance of gastric cancer cells via targeting IGF1R and BCL2.

We investigated the possible role of miR-143 in the development of cisplatin resistance in human gastric cancer cell line. miR-143 was detected by quantitative real-time PCR. Cisplatin resistance changes of cells was tested via MTT assay. Target genes of miR-143 were verified by dual-luciferase activity assay. Immunohistochemistry, immunofluorescence staining, Western blot, cell proliferation, and clonogenic and apoptosis assay were used to elucidate the mechanism of miR-143 in cisplatin resistance formation. miR-143 was downregulated in gastric cancer tissues and cell lines. It was also downregulated in cisplatin-resistant gastric cancer cell line SGC7901/cisplatin (DDP), which was concurrent with the upregulation of IGF1R and BCL2, compared with the parental SGC7901 cell line, respectively. Overexpressed miR-143 sensitized SGC7901/DDP cells to cisplatin. The luciferase activity suggested that IGF1R and BCL2 were both target genes of miR-143. Enforced miR-143 reduced its target proteins, inhibited SGC7901/DDP cells proliferation, and sensitized SGC7901/DDP cells to DDP-induced apoptosis. Our findings suggested that hsa-miR-143 could modulate cisplatin resistance of human gastric cancer cell line at least in part by targeting IGF1R and BCL2.

Inhibition of Vav3 could reverse the drug resistance of gastric cancer cells by downregulating JNK signaling pathway.

This study aims to investigate the effect and mechanism of Vav3 on the multidrug resistance of gastric cancer. Fluorescence quantitative RT-PCR and western blot assay were used to detect Vav3 and drug resistance genes in gastric cancer tissues as well as gastric cell lines such as SGC7901, SGC7901/adriamycin (ADR) and GES-1. Besides, Vav3-specific small interfering RNA (Vav3-siRNA) was applied to inhibit Vav3 in SGC7901/ADR, and SRB assay was used to determine chemosensitivity. After that, drug resistance genes and proteins in MAPK and PI3K/AKT signaling pathway were detected after Vav3-siRNA transfection. The results showed that overexpressed Vav3 was found in gastric cancer tissues and SGC7901 and SGC7901/ADR cells. Activity of SGC7901/ADR cells transfected with Vav3-siRNA combined with 5-fluorouracil/oxaliplatin was much lower than that of control groups, and MDR1/P-gp, GST-π and Bcl-2, Bax genes were significantly downregulated in Vav3-siRNA transfection group. AKT, ERK and p38 total protein and their phosphorylation levels showed no significant change in Vav3-siRNA-transfected SGC7901/ADR cells, whereas the ratio of C-Jun phosphorylation levels to total C-Jun protein was significantly downregulated. The results suggested that Vav3 may play a role in drug resistance of gastric cancer by inhibiting drug resistance genes MDR1/P-gp, GST-π and Bcl-2 through regulating the JNK signaling pathway.

The S100A4 D10V polymorphism is related to cell migration ability but not drug resistance in gastric cancer cells.

Upregulation of the metastasis-promoting S100A4 protein has been linked to tumor migration and invasion, and clinical studies have demonstrated that significant expression of S100A4 in primary tumors is indicative of poor prognosis. However, the involvement of S100A4 in the drug responsiveness of gastric cancer remains unclear. In the present study, we used gastric cancer cell lines as a model to investigate the involvement of S100A4 in drug responsiveness. We overexpressed S100A4 in AGS and SCM-1 cells, which are characterized by relatively low-level expression of endogenous S100A4, and found that this significantly enhanced cell migration but did not affect cell survival in the presence of six common anticancer drugs. Moreover, in vitro cell proliferation was unchanged. Using RNA interference, we suppressed S100A4 expression in MKN-45 and TMK-1 cells (which are characterized by high-level expression of endogenous S100A4), and found that knockdown of S100A4 markedly attenuated cell motility but did not affect cell survival in the presence of six common anticancer drugs. Further study revealed that a single nucleotide polymorphism (SNP) of S100A4 (rs1803245; c.29A>T), which substitutes an Asp residue with Val (D10V), is localized within the conserved binding surface for Annexin II. Cells overexpressing S100A4D10V showed a significant reduction in cell migration ability, but no change in cell survival, upon anticancer drug treatment. Taken together, our novel results indicate that the expression level of S100A4 does not significantly affect cell survival following anticancer drug treatment. Thus, depending on the cell context, the metastasis-promoting effects of S100A4 may not be positively correlated with anticancer drug resistance in the clinic.

Abstract 777: Efficacy of nab-paclitaxel on 5-fluorouracil (5-FU) resistant human gastric cancer cell lines

Abstract Purpose: 5-Fluorouracil (5-FU)-based combination chemotherapy is widely used for the treatment of advanced gastric cancer in Japan. Thus, the development of effective chemotherapy for 5-FU-resistant gastric cancer after 5-FU-based chemotherapy is important. Nab-paclitaxel is an albumin-bound, 130-nm particle form of paclitaxel that exhibits a higher activity and lower toxicity than solvent-based paclitaxel. In the present study, the activity of nab-paclitaxel alone or in combination with 5-FU was evaluated in four 5-FU resistant cell lines. Method: 5-FU-resistant human gastric cancer cell lines were established from parent cells (MKN-45, MKN-74, NCI-N87, and KATO-III) by continuous exposure to escalating concentrations of 5-FU over a one-year period. The sensitivities of these cell lines to chemotherapeutic agents (5-FU and nab-paclitaxel) were evaluated using the WST-8 colorimetric assay. To evaluate the effects of the combination of 5-FU and nab-paclitaxel, the combination index (CI) value was calculated using CalcuSyn 2.0 (Biosoft, Cambridge, UK). The gene expressions of secreted protein acidic and rich in cysteine (SPARC) in the parent and 5-FU-resistant cell lines were evaluated using a Whole Human Genome 44K oligo DNA microarray (Agilent Technologies, Santa Clara, CA). Results: The IC50 value of 5-FU ranged from 2.35 to 36.7 μM, and its ratio (resistant cell lines/parent cell lines) ranged from 2.6 to 15.6. The IC50 value of nab-paclitaxel ranged from 4.45 to 54.5 nM, and its ratio ranged from 1.0 to 1.2. Therefore, the 5-FU-resistant cell lines used in this study showed no signs of cross-resistance to nab-paclitaxel. The calculated CI value treated with 5-FU and nab-paclitaxel was approximately 1; therefore, nab-paclitaxel additively inhibited the cell growth when administered in combination with 5-FU. Interestingly, the combination of 5-FU and nab-paclitaxel had an additive cytotoxic activity in the 5-FU-resistant human gastric cell lines in vitro. The relative expression of SPARC mRNA in the resistant cell lines to that in the parent ones ranged from 0.86 to 1.47, and SPARC expression might be weakly correlated with the acquisition of 5-FU resistance. Conclusion: Nab-paclitaxel might be a promising agent for the treatment of 5-FU-resistant human gastric cancer when used alone or in combination with 5-FU. These results suggest that nab-paclitaxel might be a promising chemotherapy for gastric cancer patients who relapse after undergoing 5-FU-based chemotherapy. Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. Efficacy of nab-paclitaxel on 5-fluorouracil (5-FU) resistant human gastric cancer cell lines. [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 777. doi:10.1158/1538-7445.AM2014-777

Inhibition of the p38 MAPK pathway sensitizes human gastric cells to doxorubicin treatment in vitro and in vivo.

Gastric cancer is the second most common cause of cancer-related deaths worldwide. Doxorubicin-based chemotherapeutic regimes have been the mainstay of systemic treatment for disseminated gastric cancer for numerous years. However, the efficacy of doxorubicin is severely limited due to chemoresistance. Chemoresistance is a tightly regulated process, under the control of numerous signal transduction pathways. Amongst these, the mitogen-activated protein kinase (MAPK) pathway has received much attention. This study assessed whether the p38 MAPK pathway is involved in doxorubicin resistance in gastric cancer cells. Doxorubicin alone or combined with the p38 MAPK pathway inhibitor SB203580 was used to treat gastric cancer cells (SGC7901 and BGC823 lines). The effect of doxorubicin on the growth and apoptosis of gastric cancer cells in the presence or absence of SB203580 was investigated by western blot analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst staining, Annexin V-FITC/propidium iodide staining followed by flow cytometry analysis, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Next, the effects of doxorubicin and SB203580, on the sensitivity of BGC-823 cells were assessed in a tumor xenograft model. The results showed that the p38 MAPK inhibitor significantly increases gastric cancer cell sensitivity to doxorubicin. Doxorubicin in combination with SB203580 significantly reduced cell viability (P<0.01) and increased cell death (P<0.01), which may be associated with the inactivation of the p38 MAPK signaling pathway, followed by the induced expression of the pro-apoptotic protein Bax and a concomitant decrease in Bcl-2 expression. These findings suggest that p38 MAPK is involved in gastric cancer cell survival, and that the inhibition of p38 MAPK signaling can reduce the tolerance of gastric cancer cells to doxorubicin treatment.

643P - Cell Growth Inhibition of Her2 Positive Trastuzumab Resistant Gastric Cancer Cell Lines By Combined Inhibition of Pi3K/Akt/Mtor and Mapk

ABSTRACT Aim: HER2 is a key genetic driver in gastric cancer development. The anti-HER2 antibody, trastuzumab, in combination with standard chemotherapy,increases survival in HER2 positive gastric cancer. However it is still unclear why HER2 overexpressing tumours are initially and subsequently nonresponsive to trastuzumab treatment. Our aim is to investigate the possible mechanisms involved in trastuzumab resistance. Methods: A panel of human gastric and oesophago-gastric cancer cell lines(NCI-N87, KATO III, OE 19) were screened for EGFR, HER2, HER3, MAPK, PI3K/Akt/mTOR and 4EBP-1 expressions by Western Blot analysis. Cells were treated with increasing concentrations of selective inhibitors such as trastuzumab (HER2 inhibitor), erlotinib (EGFR inhibitor), MEK inhibitor (BAY-86-9766) or PIK3CA/mTOR inhibitor (GDC-0980) as a single agent and/or in combination. MTT analyses were performed to evaluate the inhibitory effect of each compound on cell growth. Results: In all cancer cells EGFR, HER2, MAPK, AKT and 4EBP1 were expressed at different degree. Although high HER2 expression levels, trastuzumab caused growth inhibition only in OE19 cancer cell line with no activity showed in KATO III and NCI-N87, suggesting a trastuzumab intrinsic resistance. The combination of MEK and PIK3CA/mTOR inhibitors in cancer cell lines was able to inhibit cell growth of NCI-N87, KATOIII and OE19 with an IC50 of 0.25 µM, 0.01 µM and 0.25 µM, respectively. The role of such therapeutic doublet in achieving cell growth inhibition was accompanied by significant reduction in the expression of AKT, pAKT, MAP, pMAP, 4EBP1 and p4EBP1 compared to control and trastuzumab treatment as revealed by Western Blot analysis. Conclusions: The results of our in vitro study suggest that in HER2 trastuzumb resistant gastric cancer cell lines the double blockade of MAP and PI3K/Akt/mTOR pathways is able to induce significant growth inhibition suggesting a relevant crosstalk between the two pathways in this setting. On the basis of these preclinical data, new potential approaches could be explored in gastric cancer HER2 patients not responding to trastuzumab. Disclosure: All authors have declared no conflicts of interest.

MiR-503 regulates cisplatin resistance of human gastric cancer cell lines by targeting IGF1R and BCL2

Background Studies have shown that the drug resistance of gastric cancer cells can be modulated by abnormal expression of microRNAs (miRNAs). We investigated the role of miR-503 in the development of cisplatin resistance in human gastric cancer cell lines. Methods MiR-503 expression was measured by quantitative real-time PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays were used to examine changes in cell viability and the drug resistance phenotype of cancer cells associated with upregulation or downregulation of the miRNA. A dual-luciferase activity assay was used to verify target genes of miR-503. Immunohistochemistry, Western blotting analysis, and a flow cytometric apoptosis assay were used to elucidate the mechanism by which miR-503 modulates drug resistance in cancer cells. Results MiR-503 was significantly downregulated in gastric cancer tissues and several gastric cancer cell lines. Additionally, downregulation of miR-503 in the cisplatin (DDP)-resistant gastric cancer cell line SGC7901/DDP was concurrent with the upregulation of insulin-like growth factor-1 receptor (IGF1R) and B-cell lymphoma 2 (BCL2) expression compared with the parental SGC7901 cell line. An in vitro drug sensitivity assay showed that overexpression of miR-503 sensitized SGC7901/DDP cells to cisplatin. The luciferase activity of reporters driven by IGF1R and BCL2 3′-untranslated regions in SGC7901/DDP cells suggested that IGF1R and BCL2 were both direct target genes of miR-503. Enforced miR-503 expression in SGC7901/DDP cells reduced expression of the target proteins, inhibited proliferation, and sensitized the cells to DDP-induced apoptosis. Conclusion Our findings suggest that hsa-miR-503 modulates cisplatin resistance of human gastric cancer cells at least in part by targeting IGF1R and BCL2.

MiR-1271 Regulates Cisplatin Resistance of Human Gastric Cancer Cell Lines by Targeting IGF1R, IRS1, mTOR, and BCL2

Numerous studies showed that drug resistance of gastric cancer cells could be modulated by the abnormal expression of microRNAs (miRNAs) which target multiple cell signaling pathways. The possible function of miR-1271 in the formation of cisplatin resistance in gastric cancer cells has been investigated in this study. miR-1271 was significantly down-regulated in gastric cancer tissues and various gastric cancer cell lines. Moreover, it was down-regulated in the cisplatin-resistant gastric cancer cell line SGC7901/cisplatin (DDP) and the down-regulation of miR-1271 in SGC7901/DPP cells was accompanied by the up-regulation of insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate 1 (IRS1) pathway-related proteins, i.e., IGF1R, IRS1, serine/threonine-protein kinase mTOR (mTOR), and the apoptosis regulator Bcl-2 (BCL2), compared with the parental SGC7901 cells. Over-expression of miR-1271 sensitized SGC7901/DDP cells to cisplatin. Changes in the luciferase activity of reporter constructs harboring the 3'-untranslated region of the above proteins in SGC7901/DDP cells suggested that IGF1R, IRS1, mTOR, and BCL2 were target genes of miR-1271. Enforced miR-1271 expression repressed the protein levels of its targets, inhibited proliferation of SGC7901/DDP cells, and sensitized SGC7901/DDP cells to DDP-induced apoptosis. Overall, on the basis of the results of our study, we proposed that miR-1271 could regulate cisplatin resistance in human gastric cancer cells, at least partially, via targeting the IGF1R/IRS1 pathway.

Gastric cancer cell adhesion to laminin enhances acquired chemotherapeutic drug resistance mediated by MGr1-Ag/37LRP.

Adhesion of cancer cells to the extracellular matrix (ECM) causes a novel acquired chemotherapeutic drug‑resistant phenotype, referred to as cell adhesion-mediated drug resistance (CAM-DR). Our previous studies suggested that the adhesion molecule MGr1-Ag/37LRP may promote multidrug resistance in gastric cancer cells. Therefore, we investigated MGr1-Ag/37LRP binding-induced adhesion, and its role in CAM-DR. Initial studies revealed that, after adhesion to the ECM, the multidrug-resistant gastric cancer cell lines SGC7901/VCR and SGC7901/ADR showed significantly higher mean adhesive cell numbers than non‑resistant SGC7901 cells. We then investigated expression of MGr1-Ag/37LRP in gastric cancer cells adhering to laminin. Western blotting, RT-PCR and dual-luciferase reporter assays showed that laminin induced MGr1-Ag/37LRP expression and activity. In vitro and in vivo assays revealed that small interfering RNA against MGr1-Ag/37LRP significantly reduced CAM-DR in SGC7901/VCR cells. In vivo and in vitro analyses revealed that binding of MGr1-Ag/37LRP decreased intracellular drug accumulation by increasing P-glycoprotein and multidrug-associated protein expression, and inhibited drug-induced apoptosis by regulating Bcl-2 and Bax expression. These results indicate that MGr1-Ag/37LRP contributes to laminin-mediated CAM-DR in gastric cancer cells, and is a potentially effective target for reversing this phenomenon in gastric cancer.

942 Epigenetic Silencing of DACH1 Induces the Invasion and Migration of Gastric Cancer by Activating TGF-β Signaling

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide and a leading cause of cancer death. Loss of DACH1 expression was found in breast, prostate, endometrial cancer, hepatocellular carcinoma and colorectal cancer. To analyze the regulation and the function of DACH1 in GC, 8 gastric cancer cell lines, 8 cases of normal mucosa and 98 cases of primary GC were employed in this study. Loss of DACH1 expression is correlated with promoter region hypermethylation and re-expression of DACH1 was induced by 5-Aza-2'-deoxyazacytidine treatment in GC cell lines. DACH1 is frequently methylated in primary GC and methylation of DACH1 is associated with reduction of DACH1 expression (p<0.01). These results suggest that DACH1 expression was regulated by promoter region hypermethylation in GC. DACH1 methylation was associated with late tumor stage (p<0.01) and lymph node metastasis (p<0.05). Re-expression of DACH1 suppressed SBE4 luciferase reporter activity and inhibited TGF-β signaling by reducing the level of phosphorylated Smad2. DACH1 inhibited epithelialmesenchymal transition (EMT) and metastasis of GC by inhibition of TGF-β signaling. The tumor size is smaller in DACH1 re-expressed BGC823 cell xenograft than in control group (p<0.01). Restoration of DACH1 expression induced G2/M phase arrest and sensitized GC cells to docetaxel. In conclusion, DACH1 is frequently methylated in human GC and methylation of DACH1 may serve as detective and prognostic markers in GC. DACH1 serve as a tumor suppresser both in vitro and in vivo, suppressed GC metastasis by inhibiting TGFβ signaling. Silencing of DACH1 expression may cause resistance of GC cells to docetaxel.