Human Gastric Cancer Xenograft Model Research Articles

ABL001, a Bispecific Antibody Targeting VEGF and DLL4, with Chemotherapy, Synergistically Inhibits Tumor Progression in Xenograft Models.

Delta-like-ligand 4 (DLL4) is a promising target to augment the effects of VEGF inhibitors. A simultaneous blockade of VEGF/VEGFR and DLL4/Notch signaling pathways leads to more potent anti-cancer effects by synergistic anti-angiogenic mechanisms in xenograft models. A bispecific antibody targeting VEGF and DLL4 (ABL001/NOV1501/TR009) demonstrates more potent in vitro and in vivo biological activity compared to VEGF or DLL4 targeting monoclonal antibodies alone and is currently being evaluated in a phase 1 clinical study of heavy chemotherapy or targeted therapy pre-treated cancer patients (ClinicalTrials.gov Identifier: NCT03292783). However, the effects of a combination of ABL001 and chemotherapy on tumor vessels and tumors are not known. Hence, the effects of ABL001, with or without paclitaxel and irinotecan were evaluated in human gastric or colon cancer xenograft models. The combination treatment synergistically inhibited tumor progression compared to each monotherapy. More tumor vessel regression and apoptotic tumor cell induction were observed in tumors treated with the combination therapy, which might be due to tumor vessel normalization. Overall, these findings suggest that the combination therapy of ABL001 with paclitaxel or irinotecan would be a better clinical strategy for the treatment of cancer patients.

Establishment of a Human Gastric Cancer Xenograft Model in Immunocompetent Mice Using the Microcarrier-6.

Gastric cancer is among the most common malignant tumors of the digestive tract. Establishing a robust and reliable animal model is the foundation for studying the pathogenesis of cancer. The present study established a mouse model of gastric carcinoma by inoculating immunocompetent mice with MKN45 cells using microcarrier. Sixty male C57BL/6 mice were randomly divided into three groups: a 2D group, an empty carrier group, and a 3D group, according to the coculture system of MKN45 and the microcarrier. The mouse models were established by hypodermic injection. Time to develop tumor, rate of tumor formation, and pathological features were observed in each group. In the 3D group, the tumorigenesis time was short, while the rate of tumor formation was high (75%). There was no detectable tumor formation in either the 2D or the empty carrier group. Both H&E and immunohistochemical staining of the tumor xenograft showed characteristic evidence of human gastric neoplasms. The present study successfully established a human gastric carcinoma model in immunocompetent mice, which provides a novel and valuable animal model for the cancer research and development of anticancer drugs.

Trastuzumab in combination with paclitaxel enhances antitumor activity by promoting apoptosis in human epidermal growth factor receptor 2-positive trastuzumab-resistant gastric cancer xenograft models.

Trastuzumab, a humanized anti-human epidermal growth factor receptor 2 antibody drug, is the first-line therapy for human epidermal growth factor receptor 2-positive breast and gastric cancer. For breast cancer, the benefit of continuous treatment with trastuzumab after it becomes refractory to first-line therapy has been demonstrated. However, it is unclear whether trastuzumab can show similar efficacy as a second-line treatment for gastric cancer. Here, we report that trastuzumab in combination with paclitaxel exhibits increased antitumor efficacy even for trastuzumab-resistant xenografted tumors. We derived the trastuzumab-resistant models from previously established human epidermal growth factor receptor 2-positive gastric cancer patient-derived cells. Human epidermal growth factor receptor 2 expression, PIK3CA mutation, and phosphatase and tensin homolog expression in these resistant models was equivalent to those in the trastuzumab-sensitive parental model, whereas cyclin-dependent kinase inhibitors, such as p16, p15, and p21, were downregulated. Trastuzumab in combination with paclitaxel enhanced antitumor activity in both the sensitive and resistant models. In the trastuzumab-sensitive model, the combination of trastuzumab and paclitaxel resulted in suppression of the AKT-p27-retinoblastoma protein pathway and induction of apoptosis. Although this combination did not suppress retinoblastoma protein phosphorylation in the trastuzumab-resistant model, it did markedly decrease epidermal growth factor receptor and human epidermal growth factor receptor 2 phosphorylation and further enhance paclitaxel-mediated apoptosis. These results suggested that trastuzumab in combination with paclitaxel can still exert more potent antitumor efficacy than each agent alone in trastuzumab-resistant models, providing evidence that trastuzumab remains beneficial in the treatment of trastuzumab-resistant tumors.

A novel tetravalent bispecific antibody targeting programmed death 1 and tyrosine-protein kinase Met for treatment of gastric cancer.

Background Redirecting T cells to tumor cells using bispecific antibodies (BsAbs) is emerging as a potent cancer therapy. The main concept of this strategy is to cross-link tumor cells and T cells by simultaneously binding to cell surface tumor-associated antigen (TAA) and the CD3ƹ chain. However, immune checkpoint programmed cell death ligand-1 (PD-L1) on tumor cells or other myeloid cells upreglulated remarkablely after the treatment of CD3-binding BsAbs, leads to the generation of suppressed microenvironment for immune evasion and tumor progression. Although this resistance could be partially reversed by anti-PD-L1 treatment, targeting two pathways through one antibody-based molecule may provide a strategic advantage over the combination of BsAbs and immune checkpoint inhibitors. Methods We developed two novel BsAbs PD-1/c-Met DVD-Ig and IgG-scFv both targeting PD-1 to restore the immune effector function of T cells and engaging them to tumor cells via binding to cellular-mesenchymal to epithelial transition factor (c-Met). Binding activities, T cell activation and proliferation were analyzed by flow cytometry. Cell Cytotoxicity and cytokine release were measured using LDH release assay and ELISA, respectively. Anti-tumor response in vivo was evaluated by generate xenograft models in NOD-SCID mice. Results These bispecific antibodies exhibited effective antitumor activity against high- and low- c-Met-expressing gastric cancer cell lines in vitro and mediated strong tumor growth inhibition in human gastric cancer xenograft models. Conclusion The engagement of the PD-1/PD-L1 blockade to c-Met-overexpressing cancer cells is a promising strategy for the treatment of gastric cancer and potentially other malignancies.

Abstract LB-107: Targeting ATR using a novel ATR inhibitor AZD6738 in human gastric cancer cells

Abstract Introduction: The DNA repair system is critical for maintaining genomic integrity. In particular, the homologous recombination (HR) repair pathway, on that repairs DNA double-strand breaks (DSBs), is directly associated with cancer development. Many cancer cells could contain mutations in genes involved in the HR pathway including BRCA1/2, ATM, ATR, and RAD51. ATR can be activated by various types of DNA damage and it initiates DNA damage-induces signaling cascade. As a result, ATR pathway components are considered promising therapeutic targets because ATR inhibition is likely to have greater deleterious effect on cancer cells. Materials and Methods: The antiproliferative activity of AZD6738 was examined in vitro using a cytotoxic assay, cell cycle analysis, and western blotting. To figure out the action mechanism of AZD6738 in gastric cancer cells, the expression of proteins which participate in DNA repair were investigated. The accumulation of DNA damage was also assessed with a DNA comet assay. These in vitro data were validated in vivo using a human gastric cancer xenograft model. Results: Human gastric cancer cell lines showed heterogeneous response to AZD6738. In AZD6738 sensitive cells, ATR inhibition leads to the accumulation of unrepaired DSBs due to dysfunctional RAD51 foci formation along with increased caspase 3-dependent cell death and S phase cell cycle arrest. Compared with sensitive cells, the activation of ATM-chk2 signaling pathway under the ATR inhibition was observed in the AZD6738 insensitive cells. It suggests that the activation of ATM-chk2 signal leads to attenuation of AZD6738 sensitivity. Furthermore, AZD6738 significantly suppressed tumor growth with increased apoptosis in vivo. Conclusion: We evaluated the anti-tumor activity of AZD6738 in gastric cancer in vitro and in vivo model. This is the first study to show that AZD6738 interferes with RAD51-mediated homologous recombination, as well as promotes cell death by inducing S cell cycle arrest and apoptosis in gastric cancer cells. Our findings can help to promote novel treatment strategies using AZD6738 in gastric cancer. Citation Format: Ahrum Min, Seock-Ah Im, Hyemin Jang, Seongyeong Kim, Miso Lee, Jungeun Kim, Kyung-Hun Lee, Sae-Won Han, Tae-Young Kim, Do-Youn Oh, Tae-You Kim, Woo-Ho Kim, Yung-Jue Bang. Targeting ATR using a novel ATR inhibitor AZD6738 in human gastric cancer cells. [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 LB-107. doi:10.1158/1538-7445.AM2015-LB-107

Superior antitumor activity of trastuzumab combined with capecitabine plus oxaliplatin in a human epidermal growth factor receptor 2-positive human gastric cancer xenograft model.

In the treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastroesophageal junction cancer, it has been reported that the combination of trastuzumab with capecitabine plus cisplatin, or with 5-fluorouracil (5-FU) plus cisplatin, significantly increased overall survival compared with chemotherapy alone (ToGA trial). In addition, adjuvant therapy with capecitabine plus oxaliplatin (XELOX) improved the survival of patients who received curative D2 gastrectomy (CLASSIC trial). However, the efficacy of the combination of trastuzumab with XELOX for patients with HER2-positive gastric cancer remains unknown. The aim of this study, was to investigate the efficacy of the combination of trastuzumab with XELOX in a HER2-positive human gastric cancer xenograft model. Combination treatment with these three agents (trastuzumab 20 mg/kg, capecitabine 359 mg/kg and oxaliplatin 10 mg/kg), was found to exhibit a significantly stronger antitumor activity in NCI-N87 xenografts compared with either trastuzumab or XELOX alone. In this model, treatment with trastuzumab alone or trastuzumab plus oxaliplatin enhanced the expression of thymidine phosphorylase (TP), a key enzyme in the generation of 5-FU from capecitabine in tumor tissues. In in vitro experiments, trastuzumab induced TP mRNA expression in NCI-N87 cells. In addition, NCI-N87 cells co-cultured with the natural killer (NK) cell line CD16(158V)/NK-92 exhibited increased expression of TP mRNA. When NCI-N87 cells were cultured with CD16(158V)/NK-92 cells in the presence of trastuzumab, the mRNA expression of cytokines reported to have the ability to induce TP was upregulated in tumor cells. Furthermore, a medium conditioned by CD16(158V)/NK-92 cells also upregulated the expression of TP mRNA in NCI-N87 cells. These results suggest that trastuzumab promotes TP expression, either by acting directly on NCI-N87 cells, or indirectly via a mechanism that includes trastuzumab-mediated interactions between NK and NCI-N87 cells. Therefore, the combination of trastuzumab with XELOX may be a potent therapy for HER2-positive gastric cancer.

Abstract LB-97: A novel anticancer bispecific antibody targeting VEGF and Dll4 inhibits tumor progression in lung and gastric cancer xenograft models

Abstract Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been used for cancer treatment. However, VEGF inhibitors alone can promote tumor invasion and metastasis by increase of intratumoral hypoxia in some preclinical models. Emerging reports regarding angiogenesis suggest Delta-like-ligand 4 (Dll4) is a promising target to augment the effects of VEGF inhibitors. Dll4 blockade inhibits tumor progression by increasing nonproductive angiogenesis whereas VEGF blockade inhibits tumor progression by decreasing angiogenesis. To evaluate the effect by inhibiting both VEGF and Dll4 on tumor angiogenesis, we have developed HD105, a novel bispecific antibody targeting VEGF and Dll4, as the format of VEGF-binding IgG linked with Dll4-binding single-chain Fv. The bispecific antibody showed potent binding affinity against VEGF (KD: 3.4 x 10-12 M) and Dll4 (KD: 4.2 x 10-8 M), which are comparable with KD values of anti-VEGF antibody (9.8 x 10-10 M) and Dll4 single targeting antibody (5.5 x 10-9 M). The HD105 bispecific antibody also inhibited interaction of Dll4 and its receptor, Notch 1 (IC50: 1 nM ± 0.2). By using in vitro cell-based assays, the HD105 effectively blocked Dll4 as well as VEGF signaling pathways in endothelial cells, resulting in downregulation of Notch intracellular domain (NICD) and VEGFR-2/extracellular-signal-regulated kinase (ERK) phosphorylation. In addition, the HD105 more efficiently inhibited tumor progression than each single-targeting antibody in A549 human lung cancer and SCH human gastric cancer xenograft models. In conclusion, the novel bispecific antibody could be potentially developed as a clinically more efficacious therapeutic antibody. Citation Format: Weon-Kyoo You, Dongheon Lee, Yu Bin Choi, Kyung Jae Kang, Dong In Kim, Jin-Hyung Ahn, Jinwon Jung, Eun-Sil Sung, Jun Seo Goo, Hyun Sook Jang, Kyung Duk Moon, Sang Hooon Lee. A novel anticancer bispecific antibody targeting VEGF and Dll4 inhibits tumor progression in lung and gastric cancer xenograft models. [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 LB-97. doi:10.1158/1538-7445.AM2014-LB-97

Effect of brachytherapy on NF-κB and VEGF in gastric carcinoma xenografts.

Iodine-125 (125I) seed irradiation can be used as an important supplementary treatment for unresectable advanced gastric cancer. However, the radiobiological mechanism underlying brachytherapy remains unclear. Therefore, we investigated the influence of continuous and low-energy 125I irradiation on the cell cycle distribution, apoptosis, expression of NF-κB and VEGF and tumor growth in a human gastric cancer xenograft model. To create an animal model of gastric cancer, SGC-7901 cells were surgically implanted into mice. The 60 mice bearing SGC-7901 gastric cancer xenografts were randomly separated into 2 groups. Sham seeds (0 mCi) were implanted into the control group (n=30); 125I seeds (0.6 mCi) were implanted into the treatment group (n=30). At 28 days after irradiation, apoptosis was detected by flow cytometry. fluorescence micrograph detected intense VEGF and NF-κB immunofluorescence in the tumor samples, and changes in NF-κB and VEGF mRNA and protein expression were assessed by real-time PCR and western blot analysis, respectively. The tumor volume and weight were measured 0-28 days after 125I seed implantation. 125I seed irradiation induced significant apoptosis and G2/M phase arrest. Reduction in the intensities of VEGF and NF-κB immunofluorescence in tumor vessels was observed after treatment. NF-κB and VEGF mRNA and protein expression levels were substantially lower in the implantation treatment group than in the control group. Consequently, 125I seed implantation inhibited cancer growth and reduced cancer volume. The present study revealed that 125I seed irradiation significantly induced apoptosis and cell cycle arrest in the human gastric cancer xenografts. 125I-induced changes in NF-κB and VEGF expression are suggested as potential mechanisms underlying effective brachytherapy.

A phase I dose escalation study to evaluate safety and tolerability of cabazitaxel (Cbz) as a single agent in patients (pts) with advanced gastric adenocarcinoma who have failed prior chemotherapy (CT) regimens (GASTANA).

141 Background: Cbz showed antitumor activity in a CT-resistant human gastric cancer xenograft model. The Phase I GASTANA study aimed to determine the safety profile of Cbz in pts with advanced gastric adenocarcinoma who failed prior CT regimens. Methods: Asian pts with metastatic gastric adenocarcinoma failing 2 prior CT regimens received Cbz (single agent, Day 1 of every 3-week cycle) in a standard 3+3 dose escalation design. Dose levels (DL) were 20 mg/m2 (DL 1), 25 mg/m2 (DL 2) and 15 mg/m2 (DL -1). Treatment continued until disease progression or unacceptable toxicity. Prophylactic G-CSF was not permitted at cycle 1. Results: Fifteen pts were evaluable for dose-limiting toxicities (DLTs) at cycle 1. All pts were heavily pretreated (median of 2 prior anticancer regimens), and 11 had prior taxane exposure.At DL 1, no DLTs occurred in any of the first 3 pts. At DL 2, 4 pts were enrolled as 1 pt discontinued prematurely, with only 1 DLT (Grade [Gr] 4 febrile neutropenia [FN]) observed. However, all 4 pts at DL 2 experienced FN, and so 3 more pts were enrolled at DL 1 to further explore safety at this lower DL. Two DLTs (Gr 4 neutropenia > 7 days) occurred in these additional 3 pts. In response, DL -1 was opened, with no DLTs observed in the 6 pts enrolled. The median numbers of cycles were 5 (DL 1), 1.5 (DL 2) and 3 (DL -1). Frequent Gr 3/4 toxicities (safety population, N = 16) included neutropenia (63%) and FN (38%). Best overall responses included 1 partial response (6.3%; DL -1) and 8 stable disease (50%). Conclusions: Due to the unexpectedly high incidence of neutropenia-related complications compared with pts with other cancer types, a further Phase II study of Cbz was put on hold pending further data in pts with gastric cancer. The frequent occurrence of neutropenic complications with Cbz in this study may be attributed in part to the heavily pretreated nature of the pts and the accumulated toxicity of prior taxane therapy. Also, prophylactic G-CSF use after cycle 1 was not mandatory in pts with dose interruptions due to neutropenia, which may have increased the rate of neutropenic complications in subsequent cycles. Clinical trial information: NCT01497964.

Effects of Exogenous Growth Hormone on Growth Hormone-Insulin-Like Growth Factor Axis of Human Gastric Cancer Cell

Aim: To study effects of recombinant human growth hormone (rhGH) on growth hormone-insulin-like growth factor axis (GH-IGFs) of human gastric cancer cell in vivo in order to reveal part mechanism of growth effects of rhGH on gastric cancer. Methods: Nude mice were randomly divided into control group, cisplatin (DDP) group, rhGH group and DDP + rhGH group after human gastric cancer xenograft model of node mice was successfully founded and drugs were used for 6 days. We investigated volume of tumor, inhibitory rate of tumor and cell cycle by slide gauge and flow cytometry. In addition, We also respectively investigated insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) of blood serum of nude mice, IGF-ImRNA, insulin-like growth factor-I receptor (IGF-IR) mRNA and IGFBP-3 mRNA of xenograft of nude mice by enzyme linked immunosorbent assay (ELISA) and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on the first day of completing use of drugs later. Results: Tumor grew obviously slowly and tumor inhibitory rate obviously rose in DDP group and DDP + rhGH group compared with control group and rhGH group (p p p < 0.05). Expressions of IGF-I mRNA and IGF-IR mRNA were not obviously different in all groups. But expression of IGFBP-3 mRNA obviously increased in rhGH group, DDP group and DDP + rhGH group compared with control group; meanwhile, expression of IGFBP-3 mRNA also obviously increased in DDP + rhGH group compared with control group, DDP group and rhGH group. Conclusion: Our results indicated rhGH in short-time use did not improve proliferation of human gastric cancer cells and its mechanism was possible that rhGH in short-time use raised simultaneously IGF-I and IGFBP-3 of blood serum and increased IGFBP-3 mRNA, but degraded ratio of IGF-I and IGFBP-3 of blood serum in human gastric cancer cells.

A Heterodimeric Fc-Based Bispecific Antibody Simultaneously Targeting VEGFR-2 and Met Exhibits Potent Antitumor Activity

Heterodimeric Fc designed by engineering the CH3 homodimeric interface of immunoglobulin G1 serves as an attractive scaffold for the generation of bispecific antibodies (bsAb) due to the favorable properties of the Fc region. In this study, we describe a heterodimeric Fc generated by substituting the conserved electrostatic interactions at the CH3 core interface with asymmetric hydrophobic interactions and introducing asymmetric, long-range electrostatic interactions at the rim of the CH3 interface. Coexpression of Fc proteins carrying the combined CH3 variant pairs in HEK293F cells produced the heterodimer, which was purified with more than 90% yield, and retained wild-type Fc biophysical properties. The heterodimeric Fc was exploited to generate a bsAb simultaneously targeting both the Met receptor tyrosine kinase and the VEGF receptor 2 (VEGFR-2), with two respective antigen-specific, single-chain variable fragments (scFv) into the N-terminus. The Met × VEGFR-2 bsAb bound concurrently to the two target antigens, efficiently inhibited the downstream signaling and tube formation stimulated by the two receptors in human endothelial cells, and exhibited more potent antitumor efficacy in MKN45 human gastric cancer xenograft models than both the parent monospecific antibody alone. Collectively, based on the newly designed heterodimeric Fc-based bsAb, our results provide the therapeutic potential of bsAb targeting both Met and VEGFR-2 simultaneously for the treatment of human cancers.

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (Kd=0.52nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC50=0.098μM, NCI-N87 IC50=0.066μM) and also displayed high oral bioavailability in mice (F=44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition=136%).

Abstract C127: TAS-116, an orally available HSP90α/β selective inhibitor: Synthesis and biological evaluation.

Abstract Background: The molecular chaperone HSP90 has long been considered a promising target for cancer therapy; however, several HSP90 inhibitors have demonstrated excessive adverse effects in clinical trials potentially involving off-target and/or HSP90-related toxicities. TAS-116 is a novel, potent and orally active HSP90α/β inhibitor discovered by chemical optimization of an initial hit compound having a 4-(1H-indol-1-yl)benzamide core structure. Here we describe the structure evolution and biological evaluation of TAS-116. Results: Hit to lead investigations of a 4-(1H-indol-1-yl)benzamide derivative were accomplished by introduction of an alkyl group at the 3-position, inserting one or two nitrogen atoms into the indole ring and ring expansion. As a consequence of chemical structure evolution, we identified novel 5, 6- and 6, 6-fused heteroaromatic rings, especially, the pyrazolopyridine system, which showed increased HSP90-binding affinity in vitro, and cell growth inhibition with HSP90 client degradation. Regarding the 4-position of pyrazolopyridine, substituted imidazoles showed the most favorable profile in vitro inhibitory activities. As for the SAR of the benzamide moiety at the 2- and 3-position, introduction of alkyl and aminoalkyl groups at the 2- or 3-position showed favorable properties, in particular for oral absorption in mice. The clinical candidate, TAS-116, which was identified as a result of these chemistry efforts, showed potent and selective HSP90α/β inhibitory activities in vitro as well as good oral bioavailability in mice, rats and dogs. With regard to potential drug-drug interactions, TAS-116 did not inhibit major cytochrome P450 enzymes in vitro at effective concentrations of TAS-116. TAS-116 demonstrated tumor shrinkage in a NCI-N87 (human gastric cancer) xenograft model without significant body weight loss by oral administration. Preparation of TAS-116 analogues was accomplished through coupling reactions with 4-substituted-pyrazolopyridines which were prepared over several steps and appropriate benzonitriles. Conclusion: A novel, potent and orally active HSP90α/β selective inhibitor, TAS-116, was discovered by rational design and synthesis from a 4-(1H-indol-1-yl)benzamide derivative. The oral clinical candidate, TAS-116, shows favorable drug-like properties and warrants clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C127. Citation Format: Takao Uno, Makoto Kitade, Satoshi Yamashita, Hiromi Oshiumi, Yuichi Kawai, Takashi Mizutani, Chihoko Yoshimura, Yasuo Kodama, Hiromi Muraoka, Kouichi Takahashi, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α/β selective inhibitor: Synthesis and biological evaluation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C127.

BAY 11-7082, a nuclear factor-κB inhibitor, induces apoptosis and S phase arrest in gastric cancer cells

Inhibitors of nuclear factor (NF)-κB pathway have shown potential anti-tumor activities. However, it is not fully elucidated in gastric cancer. Firstly, we screened the inhibitory effect of pharmacologic NF-κB inhibitors on cell viability of human gastric cancer cells via CCK-8 assay. Next, cell apoptosis, cell cycle distribution, and mitochondrial membrane potential after BAY 11-7082 treatment were detected by annexin V staining, propidium iodide staining, TUNEL, and JC-1 assays in human gastric cancer HGC-27 cells. Expression of regulatory factors for apoptosis and cell cycle were measured by western blot. Finally, human gastric cancer xenograft model was established to verify the anti-tumor effects of BAY 11-7082 in vivo. Cellular apoptosis and growth inhibition in subcutaneous tumor section were detected by TUNEL and immunohistochemistry assays. BAY 11-7082 exhibited rapid and potent anti-tumor effects on gastric cancer cells in vitro within a panel of NF-κB inhibitors. BAY 11-7082 induced rapid apoptosis in HGC-27 cells through activating the mitochondrial pathway, as well as down-regulation of Bcl-2 and up-regulation of Bax. BAY 11-7082 also induced S phase arrest through suppressing Cyclin A and CDK-2 expression. Xenograft model confirmed the anti-tumor effects of BAY 11-7082 on apoptosis induction and growth inhibition in vivo. Our results demonstrated that BAY 11-7082 presented the most rapid and potent anti-tumor effects within a panel of NF-κB inhibitors, and could induce cellular apoptosis and block cell cycle progression both in vitro and in vivo, thus providing basis for clinical application of BAY 11-7082 in gastric cancer cases.

125I seed irradiation induces up-regulation of the genes associated with apoptosis and cell cycle arrest and inhibits growth of gastric cancer xenografts

BackgroundIodine 125 (125I) seed irradiation can be used as an important supplementary treatment for unresectable advanced gastric cancer. Here, we aim to comprehensively elucidate the biological effects induced by 125I seed irradiation in human gastric cancer xenograft model by using global expression and DNA methylation analyses.MethodsThe 48 mice bearing NCI-N87 gastric cancer xenografts were randomly separated into 2 groups: sham seeds (O mCi) were implanted into the control group (n = 24); 125 l seeds (0.9 mCi) were implanted into the treatment group (n = 24). The mitotic index and apoptotic index were evaluated by quantitative morphometric analysis of the expression of proliferating cell nuclear antigen (PCNA) and in situ terminal transferase-mediated fluorescein deoxy- UTP nick end labeling (TUNEL), respectively. Global gene expression changes induced by 125I seed irradiation were analyzed by using Nimblegen Human gene expression array. DNA methylation profile in the tumors from control group was investigated with methylated DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays. The changes in the methylation status of selected genes were further investigated by using MeDIP-PCR.Results125I seed irradiation suppresses the growth of gastric cancer xenografts in nude mice. PCNA staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis contribute to the 125I-induced tumor suppression in nude mouse model. Gene expression profiles revealed that the expression levels of several hundred genes, many of which are associated with apoptosis or cell cycle arrest, including BMF, MAPK8, BNIP3, RFWD3, CDKN2B and WNT9A, were upregulated following 125I seed irradiation. Furthermore, the up-regulation of some of these genes, such as BNIP3 and WNT9A, was found to be associated with irradiation-induced DNA demethylation.ConclusionsThis study revealed that 125I seed irradiation could significantly induce the up-regulation of apoptosis- and cell cycle-related genes in human gastric cancer xenografts. And some of the up-regulation might be attributed to 125I-irradiation induced demethylation in gene promoter regions. Collectively, these findings provided evidence for the efficacy of this modality for the treatment of gastric cancer.

Abstract 4115: Depletion of unsaturated fatty acids by stearoyl-coA desaturase inhibition results in endoplasmic reticulum stress-mediated tumor cell death

Abstract Over the past few years, a more detailed picture of the importance of metabolic pathways to the maintenance and progression of human cancers has begun to emerge. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. The endoplasmic reticulum-resident Stearoyl CoA Desaturase (SCD) is the rate limiting enzyme that catalyzes conversion of saturated fatty acids to monounsaturated fatty acids, a key step in the fatty acid metabolic network. In the studies reported here, we show that inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition when tumor cells are cultured in reduced (2%) serum conditions but has little impact on cells cultured under normal (10%) serum conditions. Consistent with the observed dependence on SCD1, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. This finding was confirmed using siRNA specifically targeting SCD1. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that unsaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy. 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 4115. doi:1538-7445.AM2012-4115

Abstract 2767: AUY922, a heat shock protein 90 inhibitor, plus 5-fluorouracil inhibits tumors synergistically in gastric cancer MKN45 xenograft via down regulation of multiple pivotal growth factor receptors and oncoproteins

Abstract Heat shock protein 90 (Hsp90) is the most abundant cellular chaperone and has nearly 100 pivotal client proteins, which are commonly dysregulated in cancers. The positive rate of Hsp90beta in gastric cancer (GC) tissue was approximately 30%, and was higher than non-cancerous gastric mucosa. Hsp90 inhibitors should have great potentials in the exploration for treatment strategy in gastric cancer (GC). NYP-AUY922-AG (AUY922), kindly provided by Novartis Pharma AG, is a novel resorcinylic isoxazole amide Hsp90 inhibitor. Using 72-hour MTT colorimetric assay, we have demonstrated that AUY922 has highly active growth inhibitory effects as a single agent of IC50 in the nanomolar ranges between 8.1 ± 0.2 and 14.7 ± 0.8 nM in the gastric cancer cell line panels (except c-met-overexpressing KATO-III cells with a IC50 of 158.1 ± 5.1 nM). In the gastric cancer MKN45 xenograft model, MKN45 cells (2 × 106) were injected subcutaneously in 100 αl of Matrigel/Hanks’ balanced salt solution to the flank of nude mice. AUY922 50 mg/kgw and 100 mg/kgw intraperitoneal weekly injection (i.p.) were used. Tumor inhibitory effects of AUY922 in the MKN45 human gastric cancer xenograft model were statistically evident in a dose-dependent and time-dependent manner. Several important growth factor receptors are overexpressed in GC, namely EGFR (epidermal growth factor receptor, HER1), HER-2/neu, and c-met (hepatocyte growth factor receptor, HGF receptor), etc. By immunohistochemical staining of xenograft tumor tissues using specific antibodies, AUY922 100 mg/kgw i.p. weekly significantly down regulates multiple growth factor receptors and oncoproteins, including c-met, HER-2/neu, IGF1-R, EGFR, Akt, and notably thymidylate synthase (TS), etc. While the effects of AUY922 50 mg/kgw are less statistically significant. Up to date, 5-fluorouracil (5-FU) or oral fluoropyrimidines are the current mainstay agents for first-line treatment in gastric cancer. By combining AUY922 100 mg/kgw i.p. and 5-fluorouracil (5-FU) 40 mg/kgw i.p. weekly (wk), tumor inhibitory effects of AUY922 and 5-FU were synergistically effective. Combining AUY922 50 mg/kgw or 100 mg/kgw i.p. weekly and other relevant chemotherapeutic agents has been also actively exploring. Our data indicate that AUY922 plus 5-fluorouracil (5-FU) inhibits tumors synergistically in gastric cancer MKN45 xenograft via down regulation of multiple pivotal growth factor receptors and oncoproteins. Since phase I study of AUY922 has been completed with favorable toxicity profiles, it is highly reasonable to combine AUY922 with 5-FU or other relevant chemotherapeutic agents in future development of clinical trials for gastric cancers. 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 2767. doi:1538-7445.AM2012-2767

Curcumin Induces the Differentiation of Myeloid-Derived Suppressor Cells and Inhibits Their Interaction with Cancer Cells and Related Tumor Growth

Myeloid-derived suppressor cells (MDSC) accumulate in the spleen and tumors and contribute to tumor growth, angiogenesis, and progression. In this study, we examined the effects of curcumin on the activation and differentiation of MDSCs, their interaction with human cancer cells, and related tumor growth. Treatment with curcumin in the diet or by intraperitoneal injection significantly inhibited tumorigenicity and tumor growth, decreased the percentages of MDSCs in the spleen, blood, and tumor tissues, reduced interleukin (IL)-6 levels in the serum and tumor tissues in a human gastric cancer xenograft model and a mouse colon cancer allograft model. Curcumin treatment significantly inhibited cell proliferation and colony formation of cancer cells and decreased the secretion of murine IL-6 by MDSCs in a coculture system. Curcumin treatment inhibited the expansion of MDSCs, the activation of Stat3 and NF-κB in MDSCs, and the secretion of IL-6 by MDSCs, when MDSCs were cultured in the presence of IL-1β, or with cancer cell- or myofibroblast-conditioned medium. Furthermore, curcumin treatment polarized MDSCs toward a M1-like phenotype with an increased expression of CCR7 and decreased expression of dectin 1 in vivo and in vitro. Our results show that curcumin inhibits the accumulation of MDSCs and their interaction with cancer cells and induces the differentiation of MDSCs. The induction of MDSC differentiation and inhibition of the interaction of MDSCs with cancer cells are potential strategies for cancer prevention and therapy.

Low-Dose Docetaxel Combined with (−)-Epigallocatechin-3-Gallate Inhibits Angiogenesis and Tumor Growth in Nude Mice with Gastric Cancer Xenografts

Low-dose metronomic (LDM) chemotherapy represents a new strategy to treat solid tumors by stronger antiangiogenic activity and less side-effects, especially in combination with other antiangiogenic agents. The aim of the study is to investigate the antiangiogenic effect of docetaxel alone and combined with (-)-epigallocatechin-3-gallate (EGCG) in preclinical settings of gastric cancer. BGC-823 human gastric cancer xenograft model was used, and tumor growth, side-effects of mice were closely monitored. Expression of vascular endothelial growth factor and CD31 were observed by immunohistochemistry, and microvessel density of the tumor tissues was assessed by CD31 immunohistochemical analysis. Our results indicated that LDM docetaxel inhibited angiogenesis and growth of gastric cancer with less toxicity, and the effects were further enhanced by the concurrent administration of EGCG. Our study, for the first time, rationally demonstrated that LDM docetaxel treatment used alone or combined with EGCG is effective and safe in preclinical settings of gastric cancer. Our data suggest that LDM docetaxel used alone or combined with EGCG may be an innovative and promising therapeutic strategy in the experimental treatment of human gastric cancer.

Biomarkers for antitumor activity of bevacizumab in gastric cancer models

BackgroundBevacizumab is a humanized monoclonal antibody to human vascular endothelial cell growth factor (VEGF) and has been used for many types of cancers such as colorectal cancer, non-small cell lung cancer, breast cancer, and glioblastoma. Bevacizumab might be effective against gastric cancer, because VEGF has been reported to be involved in the development of gastric cancer as well as other cancers. On the other hand, there are no established biomarkers to predict the bevacizumab efficacy in spite of clinical needs. Therefore, we tried to identify the predictive markers for efficacy of bevacizumab in gastric cancer patients by using bevacizumab-sensitive and insensitive tumor models.MethodsNine human gastric and two colorectal cancer mouse xenografts were examined for their sensitivity to bevacizumab. We examined expression levels of angiogenic factors by ELISA, bioactivity of VEGF by phosphorylation of VEGFR2 in HUVEC after addition of tumor homogenate, tumor microvessel density by CD31-immunostaining, and polymorphisms of the VEGF gene by HybriProbe™ assay.ResultsOf the 9 human gastric cancer xenograft models used, GXF97, MKN-45, MKN-28, 4-1ST, SC-08-JCK, and SC-09-JCK were bevacizumab-sensitive, whereas SCH, SC-10-JCK, and NCI-N87 were insensitive. The sensitivity of the gastric cancer model to bevacizumab was not related to histological type or HER2 status. All tumors with high levels of VEGF were bevacizumab-sensitive except for one, SC-10-JCK, which had high levels of VEGF. The reason for the refractoriness was non-bioactivity on the phosphorylation of VEGFR2 and micro-vessel formation of VEGF, but was not explained by the VEGF allele or VEGF165b. We also examined the expression levels of other angiogenic factors in the 11 gastrointestinal tumor tissues. In the refractory models including SC-10-JCK, tumor levels of another angiogenic factor, bFGF, were relatively high. The VEGF/bFGF ratio correlated more closely with sensitivity to bevacizumab than with the VEGF level.ConclusionsVEGF levels and VEGF/bFGF ratios in tumors were related to bevacizumab sensitivity of the xenografts tested. Further clinical investigation into useful predictive markers for bevacizumab sensitivity is warranted.