Cancer stem cell-like SP cells have a high adhesion ability to the peritoneum in gastric carcinoma

Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, but the evidence to conclusively prove this hypothesis remains uncertain. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC-rich population. The aim of this study was to clarify the characterization of the SP cells in peritoneal metastasis of gastric carcinoma. Gastric cancer cell lines OCUM-2M, OCUM-2D, and OCUM-2MD3 (a daughter cell line with high potential for peritoneal metastasis) were used. We isolated SP cells from OCUM-2M and OCUM-2D using flow cytometry. Serial sorting was performed three times to enrich SP cells, and they were designated as OCUM-2M/SP and OCUM-2D/SP cells. Flow cytometric analysis showed 0.46%, 0.29%, 5.24%, 6.49%, and 11.3% of the SP cells to be found in OCUM-2M, OCUM-2D, OCUM-2MD3, OCUM-2M/SP, and OCUM-2D/SP cells, respectively. The intraperitoneal inoculation of SP cells and OCUM-2MD3 cells produced peritoneal metastasis, but parent cells did not. The adhesion ability of SP and OCUM-2MD3 cells was significantly high in comparison to that of parent cells. The expression level of adhesion molecules α2 -, α5 -, β3 -, and β5-integrin , and CD44 , was high in SP cells compared to parent cells. The expression of stemness markers, Oct3/4 and Sox2, increased in the SP-cell-injected tumors. These findings suggested that CSC-like SP cells expressing α2 -, α5 -, β3 -, and β5-integrin , and CD44 , may play an important role for peritoneal metastasis in gastric carcinoma. Oct3/4 and Sox2 may be associated with CSC in gastric cancer. ( Cancer Sci 2009)     

ICAM-2 gene therapy for peritoneal dissemination of scirrhous gastric carcinoma.

PURPOSE: Human scirrhous gastric carcinoma develops peritoneal dissemination with high frequency, and the prognosis of patients with peritoneal metastasis is poor. There have been few reports of an immunogene therapy for peritoneal dissemination. Intercellular adhesion molecule (ICAM)-2 is a second ligand of leukocyte function-associated antigen-1, which functions as a costimulatory molecule for effector cells. In the present study, we examined whether ICAM-2 transfection using adenovirus vector is effective gene therapy for peritoneal metastasis of gastric cancer. EXPERIMENTAL DESIGN: We constructed an adenovirus vector, AdICAM-2, that encodes the full-length human ICAM-2 gene under control of the cytomegalovirus promoter. This vector expresses high levels of ICAM-2 on the human gastric cancer cell line OCUM-2MD3, which has high peritoneal metastatic ability in nude mice. We investigated the antitumor effects of gene transfer of ICAM-2 using the adenovirus vector AdICAM-2 in vitro and in vivo. RESULTS: ICAM-2 expressed on OCUM-2MD3 cells by AdICAM-2 demonstrated significantly high adhesiveness to and cytotoxicity against peripheral blood mononuclear cells in vitro compared with the control adenovirus vector AdlacZ. Intratumoral injection of AdICAM-2 significantly inhibited the growth of s.c. tumor. Mice with peritoneal metastasis survived for a significantly longer time after AdICAM-2 injection, compared with injection of AdlacZ. Histopathological findings revealed that many natural killer cells infiltrated the peritoneal metastatic lesions after AdICAM-2 injection. CONCLUSIONS: These findings suggest that transduction of ICAM-2 into cancer cells enhances the adhesion and activation of natural killer cells, resulting in a reduction of peritoneal metastasis. ICAM-2 transfection using adenovirus vector might be an effective form of gene therapy for peritoneal metastasis of gastric cancer.     

Role of alpha 2 beta 1- and alpha 3 beta 1-integrin in the peritoneal implantation of scirrhous gastric carcinoma.

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.     

A synergic inhibitory-effect of combination with selective cyclooxygenase-2 inhibitor and S-1 on the peritoneal metastasis for scirrhous gastric cancer cells

An inhibitory-effect of a selective cyclooxygenase-2 (COX-2) inhibitor on peritoneal metastasis of scirrhous gastric carcinoma was investigated in vivo. Peritoneal metastasis had developed after intraperitoneal inoculation of scirrhous gastric cancer cells, OCUM-2MD3, in nude mice. COX-2 inhibitor and/or S-1 were administered orally in nude mice with peritoneal metastasis. Oral administration of COX-2 inhibitor and S-1 significantly prolonged survival rates of these nude mice, compared with either alone. These findings suggested that combining S-1 and COX-2 inhibitor administration obtain a synergistic inhibitory-effect on the peritoneal metastasis of scirrhous gastric carcinoma.     

Screening of novel biomarkers for the detection of intraperitoneally disseminated cancer cells using human cDNA microarray

In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumor (SNU-1) and that of other cell lines established from the metastasis to the peritoneal cavity (KATO-III, SNU-5, SNU-719, MKN45P, HS39). The application of a high-density cDNA microarray method made it possible to analyze the expression of approximately 21,168 genes. Our examinations of KATO-III, SNU-5, SNU-719, MKN45P, and HS39 showed that several genes were up-regulated in addition to expression of sequence tags (ESTs). The analysis revealed altered up-regulation of CD44 (cell adhesion), CEA, 14-3-3, Ubiquitin A and several kinds of ESTs in gastric cancer cells from malignant ascites. We then analyzed these gastric cancer cell lines with Northern blots and observed preferential up-regulation of these selected genes in cells prone to peritoneal dissemination. RT-PCR confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination promise to provide new insights into the study of human gastric cancer peritoneal dissemination.     

VEGF-C基因转染对人胃癌OCUM-2MD3细胞VEGF-C表达的影响

目的:探讨脂质体介导VEGF-C基因转染对人胃癌OCUM-2MD3细胞VEGF-C表达的影响。方法:双酶切pCRⅡ-VEGF-C重组质粒获得人VEGF-CcDNA片段,与真核表达载体pcDNA3.1连接构建pcDNA3.1-VEGF-C重组质粒,脂质体介导转染人胃癌OCUM-2MD3细胞并筛选,RT-PCR检测转染后VEGF-CmRNA表达水平,免疫组化法、流式细胞术检测VEGF-C蛋白表达变化。结果:经酶切鉴定,pcDNA3.1-VEGF-C重组质粒含人VEGF-CcDNA。转染组VEGF-CmRNA相对吸光度值为(0.73±0.16)%,较空载组(0.54±0.13)%表达显著上调(P＜0.01)。免疫组化和流式细胞术检测转染组VEGF-C蛋白阳性表达率分别为(52.16±6.72)%、(41.83±3.65)%,较空载组(30.33±5.04)%、(30.16±3.23)%显著增加(P＜0.01或0.05)。结论:脂质体介导VEGF-C基因转染人胃癌OCUM-2MD3细胞可显著增加VEGF-C表达水平,对探索胃癌淋巴转移机制及其治疗具有重要意义。     

ICAM-1 (Intercellular Adhesion Molecule-1) Gene Transfection Inhibits Lymph Node Metastasis by Human Gastric Cancer Cells

Lymph node metastasis is one of the prognostic factors in gastric cancer. We have previously reported that decreased intercellular adhesion molecule-1 (ICAM-1) expression on cancer cells is associated with lymph node metastasis using a gastric cancer cell. In this study, we transfected ICAM-1 gene into a gastric cancer cell line, 2MLN, and analyzed the effect on lymph node metastasis in vitro and in vivo. A significantly greater amount of peripheral blood mononuclear cells (PBMC) adhered to ICAM-1 transfected 2MLN cells, 2MLN/ICAM cells, than to 2MLN/Vector cells. The lysis of 2MLN/ICAM cells by PBMC was significantly increased compared with that of 2MLN/Vector cells. The tumor growth rate of 2MLN/ICAM cells was significantly decreased in vivo. Lymph node metastases caused by 2MLN/ICAM cells were recognized as being fewer in number and smaller, while many lymph node metastases were caused by 2MLN cells. Histologic findings showed that leukocytes were heavily infiltrated in both the 2MLN/ICAM tumors and metastatic lesions, while only a few leukocytes were observed in the lesions associated with 2MLN cells. The above findings indicate that ICAM-1 gene transduction could prove to be an effective gene therapy for lymph node metastasis of gastric cancer.     

ICAM-1 (intercellular adhesion molecule-1) gene transfection inhibits lymph node metastasis by human gastric cancer cells.

Lymph node metastasis is one of the prognostic factors in gastric cancer. We have previously reported that decreased intercellular adhesion molecule-1 (ICAM-1) expression on cancer cells is associated with lymph node metastasis using a gastric cancer cell. In this study, we transfected ICAM-1 gene into a gastric cancer cell line, 2MLN, and analyzed the effect on lymph node metastasis in vitro and in vivo. A significantly greater amount of peripheral blood mononuclear cells (PBMC) adhered to ICAM-1 transfected 2MLN cells, 2MLN / ICAM cells, than to 2MLN / Vector cells. The lysis of 2MLN / ICAM cells by PBMC was significantly increased compared with that of 2MLN / Vector cells. The tumor growth rate of 2MLN / ICAM cells was significantly decreased in vivo. Lymph node metastases caused by 2MLN / ICAM cells were recognized as being fewer in number and smaller, while many lymph node metastases were caused by 2MLN cells. Histologic findings showed that leukocytes were heavily infiltrated in both the 2MLN / ICAM tumors and metastatic lesions, while only a few leukocytes were observed in the lesions associated with 2MLN cells. The above findings indicate that ICAM-1 gene transduction could prove to be an effective gene therapy for lymph node metastasis of gastric cancer.     

PI3K/Akt signalling is required for the attachment and spreading, and growth in vivo of metastatic scirrhous gastric carcinoma

Background:

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.

Methods:

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).

Results:

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.

Conclusion:

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.     

Proteomic differential display analysis shows up-regulation of 14-3-3 sigma protein in human scirrhous-type gastric carcinoma cells

This study performed proteomic differential display analysis of human scirrhous-type gastric carcinoma (SGC) cell lines and normal gastric mucosa (NGM) tissues by using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The human SGC cell lines were OCUM-1, OCUM-2M, OCUM-2MLN, OCUM-2D, OCUM-D3, OCUM-9 and OCUM-12. Among the SGC cell lines and the NGM tissues, 28 protein spots were found whose expression levels were different from the results of 2-DE: 19 protein spots appeared higher, and 9 other protein spots appeared lower in SGCs than in NGM tissues. These spots were analysed by LC-MS/MS analysis and identified by a peptide sequence tag. Identified increased spots included elongation factor 1-beta, 14-3-3 sigma, tropomyosin alpha-4 chain, protein DJ-1, nucleoside diphosphate kinase A, elongation factor Tu and peroxiredoxin-1. Western blot analysis showed increased protein level of 14-3-3 sigma in SGCs. Although OCUM-1 and AGS (gastric cancer) showed up-regulation of 14-3-3 sigma, MiaPaca-2 (pancreatic cancer), Huh-7 (HCC) and NCI-H2052 (malignant pleural mesothelioma) showed very weak expression of 14-3-3 sigma. The up-regulation of 14-3-3 sigma may play an important role in SGC carcinogenesis and progression and may be used as a diagnostic biomarker of SGC.     

Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination

Mesothelial cell monolayers have been reported to prevent infiltration of cancer cells into the peritoneum. We have previously reported that peritoneal fibrosis induced by gastric cancer cells prior to metastatization may provide a congenial environment for peritoneal metastases. In this study, we investigated the effects of peritoneal fibroblasts on peritoneal mesothelial cell morphology. Human gastric cancer (OCUM-2MD3), peritoneal fibroblast (NF-2P) and mesothelial (MS-1) cell lines were established in our laboratory. Histology of the peritoneum was investigated following intraperitoneal inoculation of serum-free conditioned media (SF-CM) from OCUM-2MD3 cells into nude mice. SF-CM from peritoneal fibroblasts was added to monolayer-cultured mesothelial cells, and their morphology was examined by phase-contrast microscopy. This experiment was conducted in the presence and absence of neutralizing antibodies against various factors. Mesothelial cells exposed to fibroblasts proliferation became hemispherical and separated from each other, while unexposed mesothelium remained as a flat monolayer. Cultured-mesothelial cells rounded up or exhibited a fibroblast-like shape following the addition of peritoneal fibroblast SF-CM. Anti-hepatocyte growth factor (HGF) neutralizing antibody partly inhibited this effect. We suggest that soluble factors, such as HGF, produced by peritoneal fibroblasts affect the morphology of mesothelial cells in monolayers so that the resulting environment may become prone to the peritoneal dissemination of cancer cells. © 1996 Wiley-Liss, Inc.     

Suppression of peritoneal metastasis in human gastric carcinoma by enhanced immunogenicity of B7-1 transfection

B7-1, a co-stimulatory factor, has been reported to induce cytotoxic T lymphocytes (CTL). In the present study, we transfected B7-1 genes into a gastric cancer cell line (2MD3) and analyzed the effects of B7-1 transduction on peritoneal metastasis in vitro and in vivo. We revealed that mononuclear lymphocytes show significantly stronger adherence and cytotoxicity to B7-1 transfected cells (2MD3/B7) than to their parent 2MD3 cells. We also demonstrated that mice inoculated with 2MD3/B7 cells in the peritoneal cavity have a significantly better survival rate than those inoculated with 2MD3 cells (log-rank test, p<0.01). Histologic findings showed that leukocytes intensively infiltrate the 2MD3/B7 metastatic nodules, but can scarcely be observed in the nodules associated with 2MD3 cells. These findings indicate that the B7-1 may play an important role in suppressing peritoneal metastasis by the mechanism of enhanced immunogenicity, and that B7-1 gene transduction might be effective against peritoneal metastases of gastric cancer.     

CD44H Plays an Important Role in Peritoneal Dissemination of Scirrhous Gastric Cancer Cells

The role of the adhesion molecule CD44H in the peritoneal adhesion and invasion of cancer cells was assessed using cell lines with low and high peritoneal seeding ability, OCUM-2M (2M) and OCUM-2MD3 (2MD3), respectively. The in vitro binding ability to peritoneal components (mesothelial cells, fibroricetin and type I collagen) and invasive ability of 2MD3 cells were higher than those of 2M cells. The expression level of CD44H on 2MD3 cells was higher than that on 2M cells as determined by western blot analysis and flow cytometry. The adhesiveness of 2MD3 cells to hyaluronic acid, which is expressed on the surfaces of mesothelial cells, was greater than that of 2M cells. The binding ability of 2MD3 cells to mesothelial cells was inhibited in the presence of anti-CD44H monoclonal antibody, but that of 2M cells was not. These results suggested that the 2MD3 cell binding to mesothelial cells is regulated by the CD44-hyaluronic acid dependent system. The in vitro binding to submesothelial components and the invasiveness of 2MD3 cells were also inhibited in the presence of anti-CD44H antibody. The in vivo inoculation of 2MD3 cells treated with an anti-CD44H antibody resulted in a significant prolongation of survival time as compared with control mice that were inoculated with 2MD3 cells alone. In conclusion, CD44H was associated with attachment not only to hyaluronic acid on mesothelial cells, but also to peritoneal stromal components. Thus, CD44H may play an important role in cancer cell binding and invasion in the peritoneal dissemination of scirrhous gastric cancer cells.     

Bone marrow-derived stromal cells are associated with gastric cancer progression

Background:

The aim of this study was to clarify the role of bone marrow-derived stromal cells (BM-SCs) expressing CD271 in the development of gastric cancer.

Methods:

The effect of human BM-SCs on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analysed by flow cytometry. We also generated a gastric tumour model by orthotopic inoculation of OCUM-2MLN cells in mice that had received transplantation of bone marrow from the CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumour stroma was examined by immunohistochemistry.

Results:

Numerous BM-SCs infiltrated the gastric tumour microenvironment; CD271 expression was found in ∼25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, whereas migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumours, and tumour invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse compared with that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression.

Conclusions:

Bone marrow-derived stromal cells might have an important role in gastric cancer progression, and CD271-positive BM-SCs might be a useful prognostic factor for gastric cancer patients.     

Establishment of a new scirrhous gastric cancer cell line with loss of heterozygosity at E-cadherin locus

Scirrhous gastric carcinoma, characterized by carcinoma cell proliferation and infiltration with extensive fibrosis in the stroma, frequently causes peritoneal metastasis. We describe here a newly established cell line, OCUM-6, derived from ascites effusion of a scirrhous gastric cancer patient. The cells are floating and round shape, similar to other scirrhous gastric carcinoma cell lines previously reported. Histologic findings of xenografted tumor obtained from OCUM-6 cells showed medullary growth with a poorly differentiated adenocarcinoma containing signet ring cells. LOH at E-cadherin locus 16q22 was observed in the OCUM-6 cells. LOH at E-cadherin locus might be closely associated with histologic findings and metastatic process of scirrhous gastric cancer. The scirrhous gastric cancer cell line, OCUM-6, may be useful for investigation of the mechanisms of peritoneal dissemination and carcinogenesis.     

Establishment of two new scirrhous gastric cancer cell lines: analysis of factors associated with disseminated metastasis.

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.     

Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line

Background

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.

Methods

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.

Results

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).

Conclusions

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.     

Histone deacetylase inhibitor, trichostatin A, increases the chemosensitivity of anticancer drugs in gastric cancer cell lines

Epigenetic alterations of the histone acetylation play an important role in the regulation of gene expression associated with cell cycles and apoptosis that may affect the chemosensitivity of gastric carcinomas. Recently, a histone deacetylase inhibitor, trichostatin A (TSA), was proven to be a chemo-sensitizer on human erythroleukemia cells. With the aim of improving the chemotherapeutic efficacy of gastric carcinoma, the effect of TSA on the chemosensitivity of several anticancer drugs in gastric carcinoma cells was investigated. Human gastric cancer cell lines, OCUM-8 and MKN-74, and 5 anticancer drugs, 5-fluorouracil (5-FU), paclitaxel (PTX), oxaliplatin (OXA), irinotecan (SN38) and gemcitabine (GEM) were used. In both gastric cancer cell lines, a synergistic anti-proliferative effect by the combination of TSA (30 ng/ml) with 5-FU, PTX or SN38 showed a synergistic anti-proliferative effect in OCUM-8 and MKN-74 cells. TSA increases the expression of p21, p53, DAPK-1 and the DAPK-2 gene in both OCUM-8 and MKN-74 cells. In conclusion, TSA is a promising chemotherapeutical agent in combination with anticancer drugs of 5-FU, PTX and SN38 in gastric cancer cell lines. The up-regulation of p53, p21, DAPK-1 and DAPK-2 might be associated with the synergistic effect of TSA.     

Upregulation of cancer-associated myofibroblasts by TGF-β from scirrhous gastric carcinoma cells

Background:

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.

Methods:

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.

Results:

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.

Conclusion:

Transforming growth factor-β from scirrhous gastric carcinoma cells upregulates the number of myofibroblasts in CAFs.