Many kinds of solid tumour have heterogeneously a hypoxic environment. Tumour hypoxia reported to be associated with more aggressive tumour phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear.
We established a scirrhous gastric carcinoma cell line (OCUM-12) from ascites associated with scirrhous gastric carcinoma, and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) was cloned from OCUM-12 cells by continuous exposure to 1% oxygen.
Histologic findings from orthotopic tumours derived from parent OCUM-12 cells and daughter OCUM-12/Hypo cells revealed poorly differentiated adenocarcinoma with extensive fibrosis that resembled human scirrhous gastric cancer. Necrotic lesions were frequently detected in the OCUM-12 tumours but were rarely found in the OCUM-12/Hypo tumours, although both types had multiple hypoxic loci. Apoptosis rate of OCUM-12 cells was increased to 24.7% at 1% O2, whereas that of OCUM-12/Hypo was 5.6%. The OCUM-12/Hypo orthotopic models developed multiple metastases to the peritoneum and lymph nodes, but the OCUM-12 models did not. OCUM-12/Hypo cells showed epithelial-to-mesenchymal transition and high migratory and invasive activities in comparison with OCUM-12 cells. The mRNA expression levels of both E-cadherin and zonula occludens ZO-1 and ZO-2 decreased in OCUM-12/Hypo cells, and that of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, matrix metalloproteinase-1 (MMP-1), and MMP-2 were increased in OCUM-12/Hypo cells.
OCUM-12 and OCUM-12/Hypo may be useful for the elucidation of disease progression associated with scirrhous gastric cancer in the setting of chronic hypoxia.
hypoxia resistant; scirrhous gastric carcinoma; cell line; epithelial-to-mesenchymal transition
Determination of the differences between cell lines which are derived from a primary tumour and a disseminated metastatic lesion from the same patient may aid in elucidating the factors associated with disseminated metastases. We report on the establishment and characterisation of two new scirrhous gastric cancer cell lines, designated OCUM-2M and OCUM-2D, derived from a 49-year-old female. OCUM-2M was derived from a primary gastric tumour, and OCUM-2D was derived from a sample of disseminated metastasis. The two cell lines were derived from the same patient. We investigated biological differences between the two cell lines to study mechanisms involved in disseminated metastasis. The growth activity of OCUM-2D cells as determined by doubling time and tumorigenicity was greater than that of OCUM-2M cells. The level of epidermal growth factor receptor (EGFR) expression in OCUM-2D cells was about twice that of OCUM-2M cells and the growth of OCUM-2D cells was stimulated more by epidermal growth factor (EGF) than that of OCUM-2M cells. The invasive activity of OCUM-2D cells was higher than that of OCUM-2M cells and was increased after addition of transforming growth factor-beta 1 (TGF-beta 1). An increase in the number of attached and spreading cells was found following the addition of 10 ng ml-1 TGF-beta 1. These findings suggest that high growth and invasive activity may play an important role in disseminated metastasis and that EGF and TGF-beta 1, which affect the growth and invasive activity of OCUM-2D cells, might be factors associated with metastasis in scirrhous gastric carcinoma. The two cell lines OCUM-2M and OCUM-2D should be beneficial for analysing mechanisms of tumour progression.
We established a highly peritoneal-seeding cell line, OCUM-2MD3, from a poorly peritoneal-seeding cell line, OCUM-2M, of human scirrhous gastric carcinoma. The intraperitoneal inoculation of OCUM-2MD3 cells produced peritoneal dissemination in nude mice, whereas that of OCUM-2M cells did not. We then investigated the correlation between seeding potential and adhesion molecule beta 1-integrins or alpha 6 beta 4-integrin. alpha 2 beta 1- and alpha 3 beta 1-integrin expression on OCUM-2MD3 cells (91.6% and 93.6%) was increased compared with that of OCUM-2M cells (47.8% and 34.3%) by flow cytometric analysis, and the expression level of the other integrins was not different between the two cell lines. The binding ability of OCUM-2MD3 cells to matrigel, fibronectin, laminin and type I collagen was significantly increased, approximately seven times, three times, eight times, and three times greater than that of OCUM-2M cells respectively. The invasiveness of OCUM-2MD3 cells was also significantly increased 8-fold over OCUM-2M cells. The binding and invasive ability of OCUM-2MD3 cells was significantly decreased following the addition of anti-alpha 2 beta 1- and alpha 3 beta 1-integrin antibody, but not by anti-alpha 6 beta 1- and alpha 6 beta 4-integrin antibody. These results suggest that adhesiveness and invasiveness in peritoneal implantation of scirrhous gastric carcinoma might be closely associated with alpha 2 beta 1- and alpha 3 beta 1-integrin.
PI3K/Akt (PKB) pathway has been shown in several cell types to be activated by ligands to cell surface integrins, leading to the metastasis of tumour cells. The signalling pathways involved in the metastatic spread of human scirrhous gastric carcinoma cells have not been defined.
The role of the PI3K/Akt pathway in an extensive peritoneal-seeding cell line, OCUM-2MD3 and a parental cell line, OCUM-2M, was investigated by assessing in vitro adhesion and spreading assay, and in vivo peritoneal metastatic model. We also examined the correlation of PI3K/Akt pathway with integrin signals by immunoprecipitations, using cells by transfection with mutant p85 (Δp85).
Adhesiveness and spreading of OCUM-2MD3 cells on collagen type IV was significantly decreased by PI3K inhibitors and expression of mutant p85, but not by inhibitors of protein kinase C (PKC) or extracellular signal-regulated kinase (ERK). Immunoprecipitation studies indicated that the PI3K/Akt pathway was associated with integrin signalling through Src and vinculin. In an in vivo experimental metastasis model, p85 inhibition reduced peritoneal metastasis of OCUM-2MD3 cells.
PI3K/Akt signalling may be required for integrin-dependent attachment and spreading of scirrhous gastric carcinoma cells, and would be translated into generating better strategies to optimise their use in cancer clinical trials.
PI3 kinase; gastric carcinoma; adhesion; spreading: metastasis; integrin signalling
Myofibroblasts in the cancer microenvironment have recently been implicated in tumour growth and metastasis of gastric cancer. However, the mechanisms responsible for the regulation of myofibroblasts in cancer-associated fibroblasts (CAFs) remain unclear. This study was performed to clarify the mechanisms for regulation of myofibroblasts in gastric cancer microenvironment.
Two CAFs (CaF-29 and CaF-33) from the tumoural gastric wall and a normal fibroblast (NF-29) from the nontumoural gastric wall, 4 human gastric cancer cell lines from scirrhous gastric cancer (OCUM-2MD3 and OCUM-12), and non-scirrhous gastric cancer (MKN-45 and MKN-74) were used. Immunofluorescence microscopy by triple-immunofluorescence labelling (α-SMA, vimentin, and DAPI) was performed to determine the presence of α-SMA-positive myofibroblasts. Real-time RT–PCR was performed to examine α-SMA mRNA expression.
Immunofluorescence microscopy showed that the frequency of myofibroblasts in CaF-29 was greater than that in NF-29. The number of myofibroblasts in gastric fibroblasts gradually decreased with serial passages. Transforming growth factor-β (TGF-β) significantly increased the α-SMA expression level of CAFs. Conditioned medium from OCUM-2MD3 or OCUM-12 cells upregulated the α-SMA expression level of CAFs, but that from MKN-45 or MKN-74 cells did not. The α-SMA upregulation effect of conditioned medium from OCUM-2MD3 or OCUM-12 cells was significantly decreased by an anti-TGF-β antibody or Smad2 siRNA.
Transforming growth factor-β from scirrhous gastric carcinoma cells upregulates the number of myofibroblasts in CAFs.
myofibroblasts; cancer-associated fibroblasts; TGF-β; scirrhous gastric carcinoma; microenvironment; interaction
Management of peritoneal dissemination is the most critical problem in gastric cancer. This study was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA.
The effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay.
OCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl-α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001).
VPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer.
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.
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.
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.
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.
gastric cancer; chemoresistance; irinotecan; EGFR inhibitor; combination therapy
Diffuse-type gastric carcinoma is a cancer with poor prognosis that has high levels of transforming growth factor β (TGF-β) expression and thick stromal fibrosis. However, the association of TGF-β signaling with diffuse-type gastric carcinoma has not been investigated in detail.
We used a lentiviral infection system to express a dominant-negative TGF-β type II receptor (dnTβRII) or green fluorescent protein (GFP) as a control in the diffuse-type gastric carcinoma cell lines, OCUM-2MLN and OCUM-12. These infected cells and the corresponding parental control cells were subcutaneously or orthotopically injected into nude mice. Angiogenesis was inhibited by infecting cells with a lentivirus carrying the gene for angiogenic inhibitor thrombospondin-1 or by injecting mice intraperitoneally with the small-molecule angiogenic inhibitor sorafenib or with anti-vascular endothelial growth factor (VEGF) neutralizing antibody (six or eight mice per group). Expression of phospho-Smad2 and thrombospondin-1 was investigated immunologically in human gastric carcinoma tissues from 102 patients. All statistical tests were two-sided.
Expression of dnTβRII into OCUM-2MLN cells did not affect their proliferation in vitro, but it accelerated the growth of subcutaneously or orthotopically transplanted tumors in vivo (eg, for mean volume of subcutaneous tumors on day 10 relative to that on day 0: dnTβRII tumors = 3.49 and GFP tumors = 2.46, difference = 1.02, 95% confidence interval [CI] = 0.21 to 1.84; P = .003). The tumors expressing dnTβRII had higher levels of angiogenesis than those expressing GFP because of decreased thrombospondin-1 production. Similar results were obtained with OCUM-12 cells. Expression of thrombospondin-1 in the dnTβRII tumor or treatment with sorafenib or anti-VEGF antibody reduced tumor growth, whereas knockdown of thrombospondin-1 expression resulted in more accelerated growth of OCUM-2MLN tumors than of GFP tumors (eg, mean tumor volumes on day 14 relative to those on day 0: thrombospondin-1–knockdown tumors = 4.91 and GFP tumors = 3.79, difference = 1.12, 95% CI = 0.80 to 1.44; P < .001). Positive association between phosphorylated Smad2 and thrombospondin-1 immunostaining was observed in human gastric carcinoma tissues.
Disruption of TGF-β signaling in diffuse-type gastric carcinoma models appeared to accelerate tumor growth, apparently through increased tumor angiogenesis that was induced by decreased expression of thrombospondin-1.
Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor β1 (TGFβ1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFβ1 and TGFβR was evaluated by real time RT-PCR. The TGFβ1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFβ1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFβR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFβR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFβ1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFβ/TGFβR signaling.
Cancer stem cells (CSCs) are responsible for cancer progression, metastasis, and recurrence. To date, the specific markers of CSCs remain undiscovered. The aim of this study was to identify novel biomarkers of gastric CSCs for clinical diagnosis using proteomics technology. CSC-like SP cells, OCUM-12/SP cells, OCUM-2MD3/SP cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used in this study. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. Candidate proteins detected by proteomics technology were validated by immunohistochemical analysis of 300 gastric cancers. Based on the results of LC-MS/MS, eight proteins, including RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, were up-regulated in both OCUM-12/SP cells and OCUM-2MD3/SP cells when compared to their corresponding parent cells. RT-PCR analysis indicated that the expression level of RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1, and CK18 was high in OCUM-12/SP and OCUM-2MD3/SP, in compared with the control of parent OCUM-12 and OCUM-2MD3. These proteins were significantly associated with advanced invasion depth, lymph node metastasis, distant metastasis, or advanced clinical stage. RBBP6, DCTPP1, HSPA4, and ALDOA expression in particular were significantly associated with a poor prognosis in the 300 gastric cancer patients. RBBP6 was determined to be an independent prognostic factor. The motility-stimulating ability of OCUM-12/SP cells and OCUM-2MD3/SP cells was inhibited by RBBP6 siRNA. These findings might suggest that the eight proteins, RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, utilizing comparative proteomics analysis, were perceived to be potential CSC markers of gastric cancer. Of the eight candidate proteins, RBBP6 was suggested to be a promising prognostic biomarker and a therapeutic target for gastric cancer.
The intake of dietary fatty acids is highly correlated with the risk of various cancers. Linoleic acid (LA) is the most abundant polyunsaturated fat in the western diet, but the mechanism(s) by fatty acids such as LA modulate cancer cells is unclear. In this study, we examined the role of LA in various steps in gastric cancer progression.
The difference in gene expression between LA-treated and untreated OCUM-2MD3 gastric carcinoma cells was examined by mRNA differential display. The involvement of candidate genes was examined by oligo- and plasmid-mediated RNA interference. Biological functions of several of these genes were examined using in vitro assays for invasion, angiogenesis, apoptosis, cell viability, and matrix digestion. Angiogenesis in vivo was measured by CD-31 immunohistochemistry and microvessel density scoring.
LA enhanced the plasminogen activator inhibitor 1 (PAI-1) mRNA and protein expression, which are controlled by PAI-1 mRNA-binding protein. LA-stimulated invasion depended on PAI-1. LA also enhanced angiogenesis by suppression of angiostatin, also through PAI-1. LA did not alter cell growth in culture, but increased dietary LA-enhanced tumour growth in an animal model.
Our findings suggest that dietary LA impacts multiple steps in cancer invasion and angiogenesis, and that reducing LA in the diet may help slow cancer progression.
gastric carcinoma; linoleic acid; plasminogen activator inhibitor 1; angiostatin; invasion
Vascular endothelial growth factor receptor-3 (VEGFR-3) signalling mediates lymphangiogenesis and lymphatic invasion; however, the effect of VEGFR-3 inhibition on the lymph node (LN) metastasis remains unclear. The aim of this study is to clarify the benefit of a VEGFR-3 inhibitor Ki23057 for LN metastasis.
Ki23057 was administered orally to gastric cancer models created by orthotopic inoculation of diffuse-type gastric cancer cells, OCUM-2MLN. The effects of Ki23057 on lymphatic vessel invasion, lymphatic vessel density, and VEGFR-3 phosphorylation were examined by immunostaining or immunoblotting.
Ki23057 inhibited the autophosphorylation of VEGFR-3, with IC50 values of 4.3 nM in the cell-free kinase assay. Murine gastric cancer models created by the orthotopic inoculation of OCUM-2MLN cells showed the diffusely infiltrating growth and frequently developed LN metastasis. The oral administration of Ki23057 significantly (P<0.01) reduced the size of orthotopic tumours and the number of the metastatic LN in gastric cancer models. The degree of lymphatic invasion and lymphangiogenesis was significantly (P<0.05) lower in the gastric tumours treated by Ki23057. Ki23057 inhibited the phosphorylation of VEGFR-3 of lymphatic endothelial cells in gastric tumours.
The inhibition of lymphangiogenesis targeting VEGFR-3 phosphorylation is a therapeutic strategy for inhibiting LN metastasis of diffuse-type gastric cancer.
diffuse-type gastric carcinoma; lymph node metastasis; orthotopic gastric cancer model; phosphorylation inhibitor; vascular endothelial cell growth factor receptor-3
In a search for proteins involved in cancer metastasis, we analyzed proteomes of the human gastric cancer cell OCUM-2M and its metastatic subline OCUM-2MLN. We observed that aspartate aminotransferase (AAT), D-site binding protein (DBP), and anterior gradient protein 2 (AGR2) are differentially expressed in metastatic OCUM- 2MLN cells. Measurement of protein expression in clinical samples indicated that DBP and AAT are also downregulated in metastatic adenocarcinoma. Additionally, urokinase- type tissue plasminogen activator is up-regulated in OCUM-2MLN cells and also in metastatic gastric cancer samples. Collectively, these results raise a possibility that AAT, DBP and AGR2 are functionally implicated in the invasiveness of gastric cancer cells.
AAT; AGR2; DBP; gastric cancer; metastasis; proteome
To explore the mechanism of increased collagen deposition in scirrhous carcinoma of the stomach, an attempt was made to define the role of transforming growth factor beta 1 (TGF-beta 1), secreted from tumour cells, as a possible humoral factor which functions in a paracrine manner to stimulate the production of collagen in regional fibroblasts. Immunohistochemical staining revealed that tumour cells in scirrhous carcinomas were generally stained more intensively than those in other types of carcinomas. On Northern blot analysis the tumour cells established from scirrhous carcinoma (KATO-III, OCUM-1 and HSC-39) exhibited relatively strong signals compared with those from non-scirrhous carcinoma (MKN-28 and MKN-45). In the culture media of scirrhous carcinoma cells, the active form of TGF-beta 1 was detected, while in those of the non-scirrhous carcinoma cells the latent form was demonstrated by both colony and radioreceptor assays. The culture medium from KATO-III showed strong stimulating activity of collagen synthesis in fibroblasts, and this activity was partially neutralised by an anti-TGF-beta 1 antibody. These results suggest that tumour cells in scirrhous carcinoma produce more active-form TGF-beta 1 than does non-scirrhous carcinoma and thus is partially responsible for the observed enhanced collagen deposition in the region.
Completeness of cytoreduction is an independent prognostic factor following cure-intended surgery for peritoneal carcinomatosis (PC). Intraoperative detection of the minimal residual disease may aid in achieving complete cytoreduction. NV1066, a genetically-engineered herpes simplex virus carrying the transgene for green fluorescent protein (GFP), selectively infects cancer cells. NV1066-infected cancer cells express GFP that can be detected by fluorescence laparoscopy. We sought to determine the feasibility of Virally-directed Fluorescent Imaging (VFI) in the intraoperative detection of minimal residual disease following cytoreductive surgery.
Human cancer cell lines OCUM-2MD3 (gastric) and JMN (malignant Mesothelioma) were infected with NV1066 at MOIs (multiplicity of infection; ratio of viral particles to cancer cells) of 0.01, 0.1 and 1.0. Viral infectivity was determined by flow cytometry for GFP and cytotoxicity was determined by a colorimetric assay. PC was established in mice by injection of OCUM cells into the peritoneal cavity. Forty-eight hours following intraperitoneal injection of NV1066, two experienced surgeons resected all visible disease and identified mice free of disease. Five independent observers examined these mice by bright-field and fluorescent laparoscopy and documented residual disease as per the peritoneal cancer index. Selective expression of GFP in tumor tissue was evaluated by histology and PCR for the viral gene ICP0.
In vitro, NV1066 infected, expressed GFP, and killed both cell lines at all MOIs. GFP signal was detected as early as 4-6 hours following infection. GFP signal intensity of infected cells was significantly higher than the autofluorescence of normal cells (230 – 670 -logs). In vivo, macroscopically undetectable tumor nodules by gross examination and conventional bright-field laparoscopy were identified by GFP fluorescence. Following resection, 8 of 13 mice thought to be free of disease were found to have residual disease as identified by green fluorescence (mean number of observations: 5 range: 1-9). Residual disease was most frequently observed in the retroperitoneum, pelvis, peritoneal surface, and liver (inter-observer agreement 99%). Specificity of NV1066 infection to tumor nodules was confirmed by immunohistochemistry and by PCR for viral gene.
We have demonstrated that virally-directed fluorescent imaging (VFI), a novel molecular imaging technology, can be used for real-time visualization of minimal residual disease following cytoreductive surgery and can improve the completeness of cure-intended resection.
herpes simplex virus; oncolytic viral therapy; peritoneal carcinomatosis; peritonectomy; gene therapy
Gastric cancer cells frequently metastasise, partly because of their highly invasive nature. Transforming growth factor-β (TGF-β) receptor signalling is closely associated with the invasion of cancer cells. The aim of this study was to clarify the effect of a TGF-β receptor (TβR) phosphorylation inhibitor on the invasiveness of gastric cancer cells.
Four gastric cancer cell lines, including two scirrhous-type cell lines and two non-scirrhous-type cell lines, were used. A TβR type I (TβR-I) kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at an ATP-binding site of TβR-I. We investigated the expression levels of TβR and phospho-Smad2, and the effects of TGF-β in the presence or absence of Ki26894 on Smad2 phosphorylation, invasion, migration, epithelial-to-mesenchymal transition (EMT), Ras homologue gene family member A (RhoA), ZO-2, myosin, and E-cadherin expression of gastric cancer cells.
TβR-I, TβR-II, and phospho-Smad2 expressions were found in scirrhous gastric cancer cells, but not in non-scirrhous gastric cancer cells. Ki26894 decreased Smad2 phosphorylation induced by TGF-β1 in scirrhous gastric cancer cells. Transforming growth factor-β1 upregulated the invasion, migration, and EMT ability of scirrhous gastric cancer cells. Transforming growth factor-β1 significantly upregulated the activity of RhoA and myosin phosphorylation, whereas TGF-β1 decreased ZO-2 and E-cadherin expression in scirrhous gastric cancer cells. Interestingly, Ki26894 inhibited these characteristics in scirrhous gastric cancer cells. In contrast, non-scirrhous gastric cancer cells were not affected by TGF-β1 or Ki26894 treatment.
A TβR-I kinase inhibitor decreases the invasiveness and EMT of scirrhous gastric cancer cells. Ki26894 is therefore considered to be a promising therapeutic compound for the metastasis of scirrhous gastric carcinoma.
scirrhous gastric cancer; TGF-β; epithelial-to-mesenchymal transition; Smad2; phosphorylation inhibitor
Stearic acid (C18:0) is a long chain dietary saturated fatty acid that has been shown to reduce metastatic tumor burden. Based on preliminary observations and the growing evidence that visceral fat is related to metastasis and decreased survival, we hypothesized that dietary stearic acid may reduce visceral fat. Athymic nude mice, which are used in models of human breast cancer metastasis, were fed a stearic acid, linoleic acid (safflower oil), or oleic acid (corn oil) enriched diet or a low fat diet ad libitum. Total body weight did not differ significantly between dietary groups over the course of the experiment. However visceral fat was reduced by ∼70% in the stearic acid fed group compared to other diets. In contrast total body fat was only slightly reduced in the stearic acid diet fed mice when measured by dual-energy x-ray absorptiometry and quantitative magnetic resonance. Lean body mass was increased in the stearic acid fed group compared to all other groups by dual-energy x-ray absorptiometry. Dietary stearic acid significantly reduced serum glucose compared to all other diets and increased monocyte chemotactic protein-1 (MCP-1) compared to the low fat control. The low fat control diet had increased serum leptin compared to all other diets. To investigate possible mechanisms whereby stearic acid reduced visceral fat we used 3T3L1 fibroblasts/preadipocytes. Stearic acid had no direct effects on the process of differentiation or on the viability of mature adipocytes. However, unlike oleic acid and linoleic acid, stearic acid caused increased apoptosis (programmed cell death) and cytotoxicity in preadipocytes. The apoptosis was, at least in part, due to increased caspase-3 activity and was associated with decreased cellular inhibitor of apoptosis protein-2 (cIAP2) and increased Bax gene expression. In conclusion, dietary stearic acid leads to dramatically reduced visceral fat likely by causing the apoptosis of preadipocytes.
Cancer stem cells (CSCs) may be postulated mediators of the chemoresistance. This study aimed to determine an effective signal inhibitor with effects on the proliferation of CSCs in combination with anticancer drugs.
We used three gastric cancer cell lines and three side population (SP)-enriched CSC cell lines. We examined the combined effects of inhibitors against stemness signals, including c-Met inhibitor SU11274, and five anticancer drugs on the CSC proliferation and mRNA expression of chemoresistance-associated genes.
The IC50 of irinotecan in SP-enriched CSC was 10.5 times higher than parent OCUM-2M cells, whereas that of oxaliplatin, taxol, gemcitabine, and 5-fluorouracil was 2.0, 2.8, 2.0, and 1.2, respectively. The SP cell lines had higher expression levels of UGT1A1, ABCG2, and ABCB1 than their parent cell lines. There was a synergistic antiproliferative effect with a combination of SU11274 and SN38 in SP cells, but not other inhibitors. The SU11274 significantly decreased the expression of UGT1A1, but not ABCG2 and ABCB1. The SN38 plus SU11274 group more effectively suppressed in vivo tumour growth by OCUM-2M/SP cells than either group alone.
Cancer stem cells have chemoresistance to irinotecan. The c-Met inhibitor may be a promising target molecule for irinotecan-based chemotherapy of gastric cancer.
cancer stem cell; gastric cancer; side population; irinotecan; c-Met inhibitor
Overexpression of Fibroblast Growth Factor Receptor 2 (FGFR2) may be a causative factor of a number of human tumors, especially gastric tumors of the poorly differentiated type. We investigated whether monoclonal antibodies (mAbs) directed against FGFR2 can inhibit the growth of tumors in xenograft models.
We generated and characterized three mAbs that recognize different epitopes on FGFR2: GAL-FR21, GAL-FR22 and GAL-FR23. The ability of the mAbs to recognize the FGFR2IIIb and FGFR2IIIc isoforms of FGFR2 was determined, as was their ability to block binding of FGF ligands to FGFR2. The capability of the mAbs to inhibit FGF-induced FGFR2 phosphorylation and to down-modulate FGFR2 expression was also investigated. Finally, the ability of the anti-FGFR2 mAbs to inhibit tumor growth was determined by establishing xenografts of SNU-16 and OCUM-2M human gastric tumor cell lines in nude mice, treating with each mAb (0.5 – 5 mg/kg i.p. twice weekly), and monitoring tumor size.
Of the three mAbs, GAL-FR21 binds only the FGFR2IIIb isoform, whereas GAL-FR22 and GAL-FR23 bind to both the FGFR2IIIb and FGFR2IIIc forms, with binding regions respectively in the D3, D2-D3 and D1 domains of FGFR2. GAL-FR21 and GAL-FR22 blocked the binding of FGF2, FGF7 and FGF10 to FGFR2IIIb. GAL-FR21 inhibited FGF2 and FGF7 induced phosphorylation of FGFR2, and both mAbs down-modulated FGFR2 expression on SNU-16 cells. These mAbs effectively inhibited growth of established SNU-16 and OCUM-2M xenografts in mice.
Anti-FGFR2 mAbs GAL-FR21 and GAL-FR22 have potential for the treatment of gastric and other tumors.
FGFR2; FGF2; FGF7; gastric cancer; xenograft
Among the most prominent metabolic alterations in cancer cells are the increase in glucose consumption and the conversion of glucose to lactic acid via the reduction of pyruvate even in the presence of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, may provide a rationale for therapeutic strategies that inhibit tumour growth by administration of a ketogenic diet with average protein but low in carbohydrates and high in fat enriched with omega-3 fatty acids and medium-chain triglycerides (MCT).
Twenty-four female NMRI nude mice were injected subcutaneously with tumour cells of the gastric adenocarcinoma cell line 23132/87. The animals were then randomly split into two feeding groups and fed either a ketogenic diet (KD group; n = 12) or a standard diet (SD group; n = 12) ad libitum. Experiments were ended upon attainment of the target tumor volume of 600 mm3 to 700 mm3. The two diets were compared based on tumour growth and survival time (interval between tumour cell injection and attainment of target tumour volume).
The ketogenic diet was well accepted by the KD mice. The tumour growth in the KD group was significantly delayed compared to that in the SD group. Tumours in the KD group reached the target tumour volume at 34.2 ± 8.5 days versus only 23.3 ± 3.9 days in the SD group. After day 20, tumours in the KD group grew faster although the differences in mean tumour growth continued significantly. Importantly, they revealed significantly larger necrotic areas than tumours of the SD group and the areas with vital tumour cells appear to have had fewer vessels than tumours of the SD group. Viable tumour cells in the border zone surrounding the necrotic areas of tumours of both groups exhibited a glycolytic phenotype with expression of glucose transporter-1 and transketolase-like 1 enzyme.
Application of an unrestricted ketogenic diet enriched with omega-3 fatty acids and MCT delayed tumour growth in a mouse xenograft model. Further studies are needed to address the impact of this diet on other tumour-relevant functions such as invasive growth and metastasis.
In order to investigate the relationship between intratumoral vasculature and progression of gastric carcinomas and between vessel counts and survival of patients with non-early gastric carcinoma, we counted the intratumoral microvessels and compared their numbers with clinicopathological parameters, as well as with the patients' survival. Microvessels were stained with anti-CD34 monoclonal antibody before counting by microscopy (x200). In a group of 181 patients who had undergone tumour resection and were followed for more than 24 months the vessel counts for 83 patients with stage IV disease were significantly higher than those for patients with any other stage of disease. Among various clinicopathological variables, haematogenous metastasis, lymph node metastasis, peritoneal metastasis, stage IV disease and non-curative resection were more frequent in the patients with highly vascularized tumours (intratumoral vessel count > 155) than in those with less vascularized tumours. As a classification of stage IV disease such as haematogenous or peritoneal metastasis generally indicates non-curative resection, it can be considered that the development of stage IV disease is associated with the increase in tumour angiogenesis. Both univariate and multivariate analyses showed that the intratumoral vessel count was significantly predictive of overall survival, when tested as either a continuous or dichotomous variable. Cox hazards model analysis showed that the vessel count was one of the significant and independent prognostic variables. Patients with highly vascularized tumours were significantly more likely to die than those with less vascularized tumours. Assessment of tumour vasculature may therefore be important, not only for its prognostic value, but also as it may help to predict responses to angiogenesis-inhibiting agents.
The membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated.
We examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model.
Parental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system.
Our findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis.
In the present study the putative chemopreventive effect of dietary fish oil (MaxEPA) on azaserine-induced pancreatic carcinogenesis in rats was investigated. Groups of rats were maintained on a semipurified low-fat (LF; 5 wt%) diet or on semipurified high-fat (HF; 25 wt%) diets containing 5 wt% linoleic acid (LA) and including 0.0, 1.2, 2.4, 4.7, 7.1 or 9.4 wt% MaxEPA. Animals fed a HF diet developed significantly higher mean numbers of atypical acinar cell nodules (AACNs), adenomas and carcinomas than animals fed a LF diet. Dietary MaxEPA caused a significant (P < 0.01) dose-related increase in mean number of AACNs (0.5 < phi < 3.0 mm). The mean number of adenomas and carcinomas remained similar among the groups. Cell proliferation was significantly lower in AACNs from animals fed HF containing 9.4% MaxEPA in comparison with HF without MaxEPA and with LF. LA levels had increased and arachidonic acid (AA) levels had decreased in blood plasma and pancreas with increasing dietary MaxEPA. Feeding MaxEPA resulted in significant decreases in 6-keto-prostaglandin (PG) F1 alpha (P < 0.05) and PGF2 alpha (P < 0.01) in non-tumorous pancreas, whereas PGE2, PGF2 alpha and thromboxane B2 (TXB2) levels were significantly (P < 0.001) higher in pancreatic tumour tissue than in non-tumorous pancreatic tissue. It is concluded that (i) dietary MaxEPA enhances dose-relatively growth of putative preneoplastic AACNs in the pancreas of azaserine-treated rats; (ii) dietary MaxEPA inhibits the conversion of LA to AA, as well as the conversion of AA to TXB2 or PGF2 alpha in non-tumorous pancreatic tissue; (iii) the high levels of PGE2, PGF2 alpha and TXB2 in pancreatic adenocarcinomas indicate a possible role for these eicosanoids in modulation of tumour growth.
Dietary peroxisome proliferator-activated receptor (PPAR)γ ligands, linoleic acid (LA) and conjugated linoleic acid (CLA), showed anticancer effects in colorectal carcinoma cells. LA is metabolized by two pathways. Cyclooxygenase (COX)-2 produces procarcinogenic prostaglandin E2, whereas 15-lipoxygenase (LOX)-1 produces PPARγ ligands. The 15LOX-1 pathway, which is dominant in colorectal adenomas, was downregulated and inversely COX-2 was upregulated in colorectal cancer. LA and CLA inhibited peritoneal metastasis of colorectal cancer cells in nude mice. The inhibitory effect was abrogated by PPARγ antisense treatment. A continuous LA treatment provided cancer cells quiescence. These quiescent cells formed dormant nests in nude mice administrated LA. The quiescent and dormant cells showed downregulated PPARγ and upregulated nucleostemin. Thus, short-term exposure to dietary PPARγ ligands inhibits cancer metastasis, whereas consistent exposure to LA provides quiescent/dormant status with possible induction of cancer stem and/or progenitor phenotype. The complicated roles of dietary PPARγ ligands are needed to examine further.
Gastric cancer is one of the most common carcinomas in China. microRNAs, a type of non-coding RNA, are important specific regulators and are involved in numerous bioprocesses of an organism. microRNA-21 (miR-21) has been identified as the most suitable choice for further investigation because it is overexpressed in nearly all solid tumors; furthermore, it has been demonstrated that miR-21 is involved in the genesis and progression of human cancer. It has been reported that PTEN, an important tumour suppressor, is regulated by multiple miRNAs. Thus, in this study we focused on the expression and significance of miR-21 in gastric cancer tissues, and the role of miR-21 in the biological behaviour and the expression of PTEN in gastric cancer cells. Real-time PCR was used to detect miR-21 expression in gastric cancer tissues, the adjacent normal tissues, and the gastric cell lines. The gastric cancer cell line BGC-823 was transfected with pre-miR-21/miR-21 inhibitor to overexpress/downregulate miR-21. The influence of miR-21 on the biological behaviour of gastric cancer cells was evaluated using the CCK-8 kit, FCMs, the scratch healing assay and the transwell test. Western blotting and the Luciferase Reporter Assay were used to evaluate the change of PTEN expression after lowered expression of miR-21 in gastric cancer cell lines. Real-time PCR analysis indicated that miR-21 exhibited higher expression in gastric cancer tissues compared to the adjacent non-tumor tissues. miR-21 expression was significantly associated with the degree of differentiation of the tumour tissues (P=0.004), as well as local invasion and lymph node metastasis (P<0.01). After transfection, pre-miR21 BGC-823 cells grew faster than the negative and control groups (P<0.01). The reduction in miR-21 expression demonstrated a remarkable effect on the biological behaviour of gastric cancer cells (P<0.05); the pre-miR-21-transfected cells healed more quickly compared to the control cells in the scratch healing assay, whereas the transwell test indicated that cell migration in vitro was notably inhibited with the downregulation of miR-21 (P<0.05). The western blot results and Luciferase Reporter Assay demonstrated that PTEN expression was remarkably increased after miR-21 inhibition (P<0.05). microRNA-21 expression was upregulated in gastric carcinoma tissues and was significantly associated with the degree of differentiation of tumour tissues, local invasion and lymph node metastasis. Overexpression of miR-21 promoted BGC-823 cell growth, invasion and cell migration in vitro, whereas downregulation of miR-21 exhibited a stronger inhibitory effect on the biological behaviour of gastric cancer cells; additionally, miR-21 inhibition may upregulate the PTEN expression level, which indicates that PTEN may be a target gene for gastric cancer initiation and development.
gastric cancer; miR-21; biological behavior; PTEN