Antisense S-oligodeoxynucleotide Research Articles

Diabetes-associated angiotensin activation enhances liver metastasis of colon cancer

We examined the effects of hyperglycemic conditions on liver metastasis of colorectal cancer (CRC). Angiotensin (A)-II increased growth, invasion, and anti-apoptotic survival in HT29 and CT26 cells. In contrast, angiotensinogen (ATG) increased these features in HT29 cells but not in CT26 cells. HT29 cells expressed A-II type 1 receptor, chymase, and rennin, whereas CT26 cells did not express renin. Renin expression and ATG-induced cell growth, invasion, and survival induced and increased as glucose concentration increased in HT29 cells and also CT26 cells. An inhibitor of renin or chymase abrogated A-II production in HT29 cells. Reduction of hepatic ATG production by cholesterol-conjugated antisense S-oligodeoxynucleotide suppressed liver metastasis of HT29 cells. An examination of 121 CRC patients showed that diabetes in CRC cases was associated with higher blood HbA1c, higher renin and A-II concentrations in the primary tumors, and higher incidence of liver metastasis than in nondiabetic cases. These results suggest that diabetes-associated angiotensin activation enhances liver metastasis of CRC and may therefore provide a possible target for antimetastatic therapy in CRC.

CD10 enhances metastasis of colorectal cancer by abrogating the anti-tumoural effect of methionine-enkephalin in the liver

ObjectiveTo examine the role of CD10, a characteristic marker of liver metastasis of colorectal cancers (CRCs).DesignThe effect of CD10 and Met-enkephalin (MENK) in CD10-positive and -negative human CRC cells was...

Antisense Phosphorothioate Oligodeoxynucleic Acid for CD10 Suppresses Liver Metastasis of Colorectal Cancer

CD10 expression is associated with metastases of colorectal cancer (CRC). In the present study, we examined association of CD10 with liver metastasis of CRC cells to clarify the therapeutic significance of CD10. CD10-positive human colon cancer cell line, HT29 cells showed inhibition of growth, invasion and colony formation by treatment with CD10 antisense S-oligodeoxynucleotide (S-ODN). In the mouse liver metastasis mode, CD10 antisense S-ODN-treated HT29 cells made less embedded cells in the liver than control HT29 cells. Number and size of metastatic foci in nude mice liver were reduced in CD10 antisense S-ODN-treated HT29 cells. Treatment with CD10 antisense S-ODN decreased phosphorylation of ERK1/2 and EGFR in HT29 cells. Intraperitoneal administration of liposome-capsulated CD10 antisense S-ODN inhibited establishment of liver metastasis and growth of established metastasis in nude mice. These findings suggest that CD10 is associated substantially with liver metastasis of CRC cells and might be a molecular target of CRC treatment.

Conjugated linoleic acid reduced metastasized LL2 tumors in mouse peritoneum

The effect of conjugated linoleic acid (CLA) on pre-existent peritoneal metastasis was examined by mouse peritoneal metastasis models. The cell growth of LL2 mouse cancer cells was suppressed by CLA in a dose-dependent manner. CLA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor (PPAR)-gamma. C57B6 mice were inoculated with LL2 cells into their peritoneal cavity. Two weeks after inoculation, colonized peritoneal cancer foci (2.2+/-0.4 mm in diameter) were treated with CLA administrated intraperitoneally (200 or 600 pmol/mouse, twice a week). CLA treatment decreased the number of peritoneal tumors: 8.7+/-0.6, 5.7+/-0.6, and 2.3+/-0.6 in untreated, 200 pmol/mouse CLA, and 600 pmol/mouse CLA groups, respectively (P<0.0001). CLA treatment decreased the size of peritoneal tumors: 3.7+/-1.5, 1.3+/-0.5, and 1.0+/-0.4 mm in untreated, 200 pmol/mouse CLA, and 600 pmol/mouse CLA groups, respectively (P<0.0001). In CLA-treated tumors, proliferating cells were decreased (P<0.0001), whereas apoptotic cells were increased (P=0.0010). CLA-treated LL2 tumors showed decrease of PPARgamma and EGFR proteins and increase of BAX protein in comparison with untreated tumors. These findings suggest that CLA possesses anti-tumor capability to peritoneal metastasis.

Peritoneal metastasis inhibition by linoleic acid with activation of PPARγ in human gastrointestinal cancer cells

The effect on peritoneal metastasis of linoleic acid (LA) was examined using in vitro treatment of cancer cells and mouse peritoneal metastasis models. Firstly, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by LA in a dose-dependent manner with increment of apoptosis. LA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor gamma (PPARgamma) or 15-lipoxygenase-1, which converts LA to PPARgamma ligands. LA significantly inhibited invasion into type-IV collagen-coated membrane of MKN28 and Colo320 cells (p<0.05). BALB/c nu/nu mice inoculated with MKN28 and Colo320 cells into their peritoneal cavities were administrated with LA intraperitoneally (weekly, four times). The LA treatment significantly diminished the number of metastatic foci of both cells in the peritoneal cavity (p<0.05). Protein production in MKN28 and Colo320 cells treated with LA showed a decrease of epidermal growth factor receptor and an increase of Bax. These findings suggest that LA inhibits invasion and metastasis of human gastric and colon cancer cells by nondietary administration.

Conjugated linoleic acid inhibits peritoneal metastasis in human gastrointestinal cancer cells

The effect of conjugated linoleic acid (CLA) on peritoneal metastasis was examined by in vitro treatment of cancer cells and mouse peritoneal metastasis models. First, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by CLA in a dose-dependent manner with an increment in apoptosis. CLA significantly inhibited invasion into type IV collagen-coated membrane of MKN28 and Colo320 cells (p < 0.05). CLA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor (PPAR)-gamma in both cell lines. BALB/c nu-nu mice were inoculated with MKN28 and Colo320 cells into their peritoneal cavity, and administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did not affect food intake or weight gain of mice. CLA treatment significantly decreased metastatic foci of both cells in the peritoneal cavity (p < 0.005). Survival rate in mice inoculated with MKN28 or Colo320 cells was significantly recovered by CLA treatment (p = 0.0025 and 0.0052, respectively). Protein production in MKN28 and Colo320 cells treated with CLA showed a decrease in epidermal growth factor receptor and transforming growth factor-alpha and an increase in Bax. These findings suggest that CLA inhibits metastasis of human gastric and colon cancer cells.

Association of Expression of Receptor for Advanced Glycation End Products and Invasive Activity of Oral Squamous Cell Carcinoma

Objectives: The receptor for advanced glycation end products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. Nevertheless, the involvement of RAGE in the development and progression of oral squamous cell carcinomas has not been elucidated. This study investigated the expression of RAGE in ten oral squamous cell carcinoma cell lines including primary and metastatic cell lines and its association with invasion and metastasis. Methods: Reverse transcriptase-polymerase chain reaction, antisense phosphorothioate (S)-oligodeoxynucleotide assay, preparation of antibody, immunohistochemical staining, immunoblot analysis, migration assay, in vitro invasion assay, and wound-healing assay were used. Results: RAGE protein expression of metastatic cancer cells treated with RAGE antisense S-oligodeoxynucleotide was significantly reduced compared to that of sense S-oligodeoxynucleotide-treated cells. The migration assay showed that invasive activity was significantly reduced in metastatic cancer cells treated with RAGE antisense S-oligodeoxynucleotide. Similarly, during invasion assays, numbers of invading cells were also reduced with the addition of RAGE antisense S-oligodeoxynucleotide-treated cells. A wound-healing assay showed that only a few RAGE antisense S-oligodeoxynucleotide-treated cancer cells migrated into the scraped area, whereas sense S-oligodeoxynucleotide-treated cells showed many budding nests in the scraped area of the metastatic cell lines. Immunohistochemically, oral squamous cell carcinoma cells in the tumour mesenchymal border were often immunopositive, whereas basal cells in the normal mucosa were scarcely positive. Conclusions: These results suggest that RAGE expression appears to be closely associated with the invasiveness of oral squamous cell carcinoma and represents a promising candidate for assessing the future therapeutic potential in treating patients with oral carcinoma.

Heme oxygenase-1 accelerates protumoral effects of nitric oxide in cancer cells

We examined the biological effects of nitric oxide (NO) and its mediator, heme oxygenase-1 (HO-1), in cancer. Urogenital cancer cell lines, SKRC, T24 and DU145, were treated with various concentrations of sodium nitroprusside (SNP), a NO donor. The medium nitrite concentration was exponentially increased according to the concentration of SNP. Cell growth inhibition by NO was observed only at high nitrite concentrations (>20 microM) in DU145 and T24 cells. Nitrite did not inhibit the growth of SKRC cells at any of the concentrations used. Doxorubicin (DXR) inhibited cell growth in the three cell lines, whereas growth inhibition recovered in the presence of <10 microM nitrite. The recovery of DXR-induced growth inhibition was closely associated with an increase in Bcl-2 in the presence of <10 microM nitrite. Vascular endothelial growth factor (VEGF) secretion was also increased in the presence of <10 and <20 microM nitrite, respectively, in DU145 and SKRC or T24 cells. The expression of HO-1 was associated with sensitivity to NO-induced growth inhibition at constitutive levels, and was induced by SNP treatment. HO-1 inhibition by HO-1 antisense S-oligodeoxynucleotide treatment increased NO-induced growth inhibition, and decreased Bcl-2 expression or VEGF secretion in the three cell lines. These findings suggest that the NO/HO-1 system has protumoral effects.

Depletion of Tumor-Infiltrating Macrophages Is Associated with Amphoterin Expression in Colon Cancer

Macrophage infiltration into colon cancer and amphoterin expression in cancer cells was examined in 42 human colon cancers invading the subserosa. The mean number of infiltrating macrophages was significantly higher in Dukes’ B cases than in Dukes’ C cases (p = 0.0065). Tumors with few infiltrating macrophages (macrophage depletion) were significantly more frequent in Dukes’ C cases than in Dukes’ B cases (p = 0.0014). No Dukes’ C cases with relevant macrophage infiltration showed macrophage-cancer cell contact, whereas 5 Dukes’ B cases showed such contact (p < 0.0001). In human colon cancer cells implanted in the cecum of nude mice, KM12SM (highly metastatic) tumors yielded less macrophage infiltration and more liver metastases than KM12C (low risk of metastasis) tumors (14 ± 3 vs. 78 ± 32 and 24 ± 6 vs. 5 ± 3 per liver, respectively). Amphoterin expression was detected at high frequency in both Dukes’ B and C cases (p = 0.0684). In macrophage-depleted cases, amphoterin expression was significantly higher than that in non-depleted cases (p = 0.0015). To confirm biological effects of amphoterin on macrophages, an infiltration assay using the cell-layered Boyden chamber was done. Infiltration of PMA-treated U937 monocytes through the KM12SM cell layer was increased by pretreatment of KM12SM cells with amphoterin antisense S-oligodeoxynucleotide exposure. Moreover, extracted amphoterin inhibited PMA-U937 monocyte infiltration in a dose-dependent manner. Thus, amphoterin may play an important role in the inhibition of macrophage infiltration into colon cancer.

Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer

Amphoterin, the major product of the high mobility group-1 gene, is a ligand associated with cancer invasion and metastasis through activation of the receptor for advanced glycation end products (RAGE). Expression of amphoterin and RAGE was examined in prostatectomy specimens from 40 patients with pT3 prostate cancer (18 non-metastatic and 22 metastatic) preoperatively treated with lutenizing hormone-releasing hormone (LH-RH) agonist. Amphoterin expression was detected in tumor cells of 6 (27%) metastatic and 0 non-metastatic cases (p<0.0001). Amphoterin was also detected in prostatic stromal cells of 14 (63%) metastatic cases and 2 (11%) non-metastatic cases (p=0.0010). RAGE production was detected in cancer cells of 16 (73%) metastatic and 6 (33%) non-metastatic cases (p=0.0244). A total of 2 (22%) non-metastatic and 16 (73%) metastatic cases showed co-expression of amphoterin and RAGE in tumor cells or in tumor cells and stromal cells (p=0.0001). The in vitro invasive capacity of PC-3, a prostatic cancer cell line that co-expressed amphoterin and RAGE, was suppressed by treatment with amphoterin antisense S-oligodeoxynucleotide (ODN). Primary cultured human prostatic stromal cells secreted no amphoterin; however, amphoterin secretion was induced by androgen deprivation. The conditioned medium of human prostatic stromal cells deprived of androgen recovered the in vitro invasive capacity of PC-3 cells suppressed by amphoterin antisense S-ODN. These results suggest that androgen deprivation provides a paracrine interaction between cancer and stromal cells through the RAGE-amphoterin system in advanced prostate cancer.

Contribution of eIF-4E inhibition to the expression and activity of heparanase in human colon adenocarcinoma cell line: LS-174T.

Heparanase degrades heparan sulfate proteoglycans (HSPGs) and is a critical mediator of tumor metastasis and angiogenesis. Recently, it has been cloned as a single gene family and found to be a potential target for antimetastasis drugs. However, the molecular basis for the regulation of heparanase expression is still not quite clear. The aim of this study was to determine whether the expression of eukaryotic initiation factor 4E (eIF-4E) correlated with the heparanase level in tumor cells and to explore the correlation between heparanase expression and metastatic potential of LS-174T cells. A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA was introduced into LS-174T cells by lipid-mediated DNA-transfection. eIF-4E protein and mRNA levels were detected by Western blot analysis and RT-PCR, respectively. Heparanase activity was defined as the ability to degrade high molecular weight (40-100 kDa) radiolabeled HS (heparan sulfate) substrate into low molecular weight (5-15 kDa) HS fragments that could be differentiated by gel filtration chromatography. The invasive potential of tumor cell in vitro was observed by using a Matrigel invasion assay system. The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels. As a result, the expression and activity of heparanase were effectively retarded and the decreased activity of heparanase resulted in the decreased invasive potential of LS-174T. eIF-4E is involved in the regulation of heparanase production in colon adenocarcinoma cell line LS-174T, and its critical function makes it a particularly interesting target for heparanase regulation. This targeting strategy in antisense chemistry may have practical applications in experimental or clinical anti-metastatic gene therapy of human colorectal carcinoma.

Expression of receptors for advanced glycation end-products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer.

The receptor for advanced glycation end-products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S-oligodeoxynucleotide was nine times less than that of sense S-oligodeoxynucleotide-treated cells. Growth of cells under RAGE antisense S-oligodeoxynucleotide treatment was not different from that seen under sense S-oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non-RAGE-expressing cell line). RAGE antisense S-oligodeoxynucleotide treatment suppressed the invasive activity of RAGE-positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen-coated membrane under RAGE antisense S-oligodeoxynucleotide treatment was significantly lower than under RAGE sense S-oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S-oligodeoxynucleotide-treated RAGE-negative MKN45 cells showed no difference. A wound-healing assay showed that no RAGE antisense S-oligodeoxynucleotide-treated MKN28 cells migrated into the scraped area, whereas sense S-oligodeoxynucleotide-treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE-positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE-positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer.

Expression and reversion of drug resistance-and apoptosis-related genes of a DDP-resistant lung adenocarcinoma cell line

To study the expression of drug resistance- and apoptosis-related genes of A549DDP cells as compared to the parental cell line A549, and its reversion by antisense s-oligodeoxynucleotide (S-ODN) of the differentially expressed genes.Sense and antisense S-ODN were transferred into A549DDP cells by lipofectin. Expression of genes related to drug resistance and apoptosis was examined by RT-PCR, immunocytochemistry and flow cytometry. Apoptosis was identified by DNA electrophoresis and TUNEL, and cell growth by MTT uptake.The expression of bcl-2 was positive and that of MRP at mRNA and protein levels was increased in A549DDP cells compared to A549 cells. MDR1, c-myc and TOPO II were similarly expressed in the two cell lines. Both cell lines were negative for c-erbB-2 expression. In A549DDP cells, the expression of bcl-2 and MRP was significantly inhibited by respective antisense S-ODN. Antisense S-ODN could also significantly inhibit proliferation of A549DDP cells, and promote cell apoptosis by reducing its resistance to cisplatin.Bcl-2 and MRP genes are responsible for the induced resistance of A549DDP cells to cisplatin.

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts.

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

Uni-axial cyclic stretch induces c-src activation and translocation in human endothelial cells via SA channel activation

The kinase activity of c-src increased and peaked at 15 min after an application of uni-axial cyclic stretch in HUVECs followed by a translocation of c-src to Triton-insoluble fraction. Suppression of c-src by an antisense S-oligodeoxynucleotide inhibited the stretch-induced tyrosine phosphorylation and morphological changes. The stretch-induced increase in c-src activity was inhibited by FK506, a specific inhibitor for calcineurin, by Gd 3+, a blocker for stretch activated channels, and by the extracellular Ca 2+ depletion suggesting the involvement of SA channels. These results strongly suggest c-src plays an important role in the downstream of SA channel activation followed by the morphological changes.