Protumoral roles of melanoma inhibitory activity 2 in oral squamous cell carcinoma.

BACKGROUND: The role of melanoma inhibitory activity 2 (MIA2) was examined in human oral squamous cell carcinoma (OSCC). METHODS: MIA2 role was examined by immunohistochemistry of human OSCCs and knockdown studies using human 3 OSCC cell lines with MIA2 expression. RESULTS: MIA2 expression was observed in 62 (66.7%) of 93 OSCCs and was associated with tumour expansion and nodal metastasis. Melanoma inhibitory activity 2 expression was inversely correlated with intratumoral infiltration of lymphocytes. Invasion and anti-apoptotic survival were reduced by MIA2 knockdown in HSC3 cells. MOLT-3 lymphocytes infiltrating the HSC3 cell layer was enhanced by MIA2 knockdown or MIA2 depletion with the antibody. In HSC3 cells, MIA2 knockdown decreased the expressions of vascular endothelial growth factor (VEGF), VEGF-C, and VEGF-D. The downregulation of VEGF-C and -D was caused by inhibition of p38 and extracellular signal-regulated kinase (ERK)1/2, respectively. Melanoma inhibitory activity 2 was co-precipitated with integrin α4 andα5 in HSC3 cells. Integrin α4 knockdown decreased p38 phosphorylation and increased apoptosis, whereas integrin α5 knockdown decreased c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis. Inhibition of JNK decreased apoptosis in the HSC3 cells. CONCLUSION: These findings suggest that the roles of MIA2 might be based on the variety of the integrins and the subtypes of mitogen-activated protein kinase.

Downregulation of miR-126 induces angiogenesis and lymphangiogenesis by activation of VEGF-A in oral cancer.

BACKGROUND: MicroRNA (miRNA)-126 (miR-126) is an endothelial-specific miRNA located within intron 7 of epidermal growth factor-like domain 7 (EGFL7). However, the role of miR-126 in cancer is controversial. METHODS: We examined the function of miR-126 in oral squamous cell carcinoma (OSCC) cells. Furthermore, a series of 118 cases with OSCC were evaluated for the expression levels of miR-126. RESULTS: MicroRNA-126 (miR-126) was associated with cell growth and regulation of vascular endothelial growth factor-A activity, and demethylation treatment increased expression levels of miR-126 and EGFL7 in OSCC cells. A significant association was found between miR-126 expression and tumour progression, nodal metastasis, vessel density, or poor prognosis in OSCC cases. In the multivariate analysis, decreased miR-126 expression was strongly correlated with disease-free survival. CONCLUSION: The present results suggest that miR-126 might be a useful diagnostic and therapeutic target in OSCC.

A 5-HT(4)-receptor activation-induced neural plasticity enhances in vivo reconstructs of enteric nerve circuit insult.

BACKGROUND: It was recently reported that some 5-HT(4)-receptor agonists increased neuronal numbers and length of neurites in enteric neurons developing in vitro from immunoselected neural crest-derived precursors. We aimed to explore a novel approach in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex. METHODS: The neural circuit insult was performed in guinea pigs by rectal transection and subsequent end-to-end one layer anastomosis. A 5-HT(4)-receptor agonist, mosapride citrate (10-100 micromol L(-1)) (applied for a patent) was applied locally at the anastomotic site. KEY RESULTS: Mosapride promoted the regeneration of the neural circuit in the impaired myenteric plexus and the recovery of the defecation reflex in the distal gut. Furthermore, mosapride generated neurofilament (NF)-, 5-HT(4)-receptor- and 5-bromo-2'-deoxyuridine (BrdU)-positive cells and surprisingly formed neural network in the newly formed granulation tissue at the anastomotic site 2 weeks after enteric nerve circuit insult. Possible neural stem cell markers, anti-distal less homeobox 2 (DLX2)- and p75-positive and NF-positive cells increased during the same time period. All actions by mosapride were inhibited by the specific 5-HT(4)-receptor antagonist, GR113808 (10 micromol L(-1)). CONCLUSIONS & INFERENCES: These results indicate that activation of enteric neural 5-HT(4)-receptors promotes reconstruction of an enteric neural circuit leading to the recovery of the defecation reflex in the distal gut, and that this reconstruction involves possibly neural stem cells. These findings indicate that treatment with 5-HT(4) agonists could be a novel therapy for generating new enteric neurons to rescue aganglionic gut disorders.

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

OBJECTIVE: To examine the role of CD10, a characteristic marker of liver metastasis of colorectal cancers (CRCs). DESIGN: The effect of CD10 and Met-enkephalin (MENK) in CD10-positive and -negative human CRC cells was investigated under in vitro and in vivo conditions. Human CRC samples were examined. MAIN OUTCOME MEASURE: CD10-positive and CD10-knockdown HT29 cells and CD10-negative and CD10-transfected Colo320 cells in nude mice were treated with MENK and/or the CD10 inhibitor (thiorphan). Intracellular signalling of MENK and delta-opioid receptor (DOR) was examined by immunoblotting. RESULTS: MENK inhibited the growth, invasion and survival of CRC cells following thiorphan-induced CD10 inactivation. Thiorphan suppressed liver metastasis of CD10-positive CRC cells. Inoculation of mice with CRC cells induced MENK expression in the liver. Inhibition of hepatic MENK expression by cholesterol-conjugated antisense S-oligodeoxynucleotide increased liver metastasis of CRC cells even when the cells did not express CD10. DOR activation by MENK decreased the phosphorylation of epidermal growth factor receptor and extracellular signal-regulated kinase and increased p38-dependent apoptosis. Nitric oxide was found to induce DOR expression in CRC cells. Co-treatment with thiorphan and a nitric oxide donor had a marked anti-tumour effect on liver metastasis of HT29 cells. Of 68 CRC patients, 19 (28%) showed CD10 expression, which was dependent on the extent of liver metastasis. MENK concentration in metastasis-positive human liver was higher than that in the normal liver. CONCLUSION: CD10 expression in CRC cells abrogates the anti-tumour effect of hepatic MENK by degrading it, which enhances liver metastasis of CD10-positive CRC cells.

Co-treatment with deoxycholic acid and azoxymethane accelerates secretion of HMGB1 in IEC6 intestinal epithelial cells.

OBJECTIVES: High-mobility group box 1 (HMGB1) is a nuclear protein that acts as a ligand of the receptor for advanced glycation end products (RAGE) and its expression enhances progression of cancer. However, the mechanism underlying HMGB1 secretion is still unclear. In this study, we examined the effect of deoxycholic acid (DCA), a promoter of colon carcinogenesis, on HMGB1 secretion. MATERIALS AND METHODS: We used an in vitro transformation model comprised of IEC6 intestinal epithelial cells treated with azoxymethane (AOM) and/or DCA. HMGB1 expression and secretion were examined by Western and Northern blot analyses, and ELISA. Intracellular translocation of HMGB1 was examined by protein fractionation. RESULTS: AOM + DCA-treated IEC6 cells showed upregulation of HMGB1 mRNA expression and increased level of HMGB1 protein in culture medium, but decreased level of HMGB1 protein in the nucleus. AOM + DCA treatment increased level of histone H4 acetylation, which induced translocation of HMGB1 from the nucleus to the cytoplasm and increased HMGB1 secretion. Leptomycin B inhibited extranuclear translocation and secretion of the HMGB1 protein. CONCLUSION: These findings suggest that DCA affects intracellular localization and secretion of HMGB1.

Reg IV enhances peritoneal metastasis in gastric carcinomas.

OBJECTIVES: The role of Regenerating (Reg) IV on peritoneal metastasis was examined in gastric cancer using. MATERIAL AND METHODS: Reg IV-transfected human gastric cancer cells (MKN28-R1, MKN28-R2, TMK1-R1), control transfectants (MKN28-R0, TMK1-R0), and REG4-knocked down MKN45 cells were examined in in vitro and in nude mice peritoneal metastasis models. RESULTS AND DISCUSSION: Increase of expression and secretion of Reg IV, and levels of BCL-2, BCL-XL,survivin, phosphorylated AKT, and phosphorylated EGFR, and decrease of nitric oxide-induced apoptosis were found in Reg IV-transfectants, whereas those were abrogated in the knockdown cells. In mice models, increased number and size of peritoneal tumors and decreased apoptosis were found in Reg IV-transfectants, whereas those were abrogated by the knockdown cells. Mice survivals were worsened in Reg IV-transfectants-inoculated mice, but were improved in Reg IV-knockdown cell-inoculated mice. Levels of Reg IV protein in peritoneal lavage fluids increased in Reg IV-transfectants inoculated mice, but decreased in Reg IV-knockdown cell inoculated mice. In metastasized human gastric cancers, Reg IV positivity in peritoneum-metastasis cases was higher than those in negative cases. Reg IV was detected in peritoneal lavage fluids from human gastric cancer patients, in whose lavages keratin mRNA was detected by reverse transcriptase-polymerase chain reaction. Collectively, Reg IV might accelerate peritoneal metastasis in gastric cancer. Reg IV in lavage fluids might be a good marker for peritoneal metastasis.

Reg IV expression is associated with cell growth and prognosis of adenoid cystic carcinoma in the salivary gland.

AIMS: Regenerating islet-derived family, member 4 (Reg IV) is associated with the progression of various cancers. The aim was to examine Reg IV expression in adenoid cystic carcinomas (ACCs) in salivary glands. METHODS AND RESULTS: Reg IV expression was detected by immunohistochemistry and compared with clinicopathological parameters. Expression of phosphorylated epidermal growth factor receptor (pEGFR), phosphorylated AKT (pAKT) and MUC2 was examined by immunohistochemistry. Reg IV function was assessed with Reg IV antisense S-oligodeoxynucleotides (AS) in ACC3 human ACC cells. Reg IV was expressed by salivary duct epithelia and acinus myoepithelia, but not in squamous epithelia. Reg IV expression was found in 41% (17/41) of ACCs, but in none of 40 oral squamous cell carcinomas (OSCCs) and was associated with nodal metastasis (P = 0.047) and poor prognosis (P = 0.012) in ACCs. Reg IV expression was associated with pEGFR (14/17, 82%) in Reg IV+ ACCs, but had no relationship with pAKT or MUC2 expression in ACCs. Cell growth was inhibited by AS treatment in Reg IV+ ACC3 cells, but not in HSC-4 OSCC cells, whereas in vitro invasion of neither cell types was affected by AS treatment. CONCLUSIONS: These results suggest that Reg IV might accelerate cell growth and disease progression of ACCs.

Reversal of expression of 15-lipoxygenase-1 to cyclooxygenase-2 is associated with development of colonic cancer.

AIMS: Two different pathways of linoleic acid (LA) metabolism have opposite effects on the development of colonic cancer: a protumoral prostaglandin cascade metabolized by cyclooxygenase (COX)-2, and an antitumoral peroxisome proliferator-activated receptor (PPAR)-gamma ligands metabolized by 15-lipooxygenase (LOX)-1. The aim was to examine the switching of the two LA metabolic pathways in colonic adenomas and carcinomas. MATERIALS AND METHODS: The expression of 15LOX-1 mRNA and COX-2 protein was examined in 54 adenomas, 21 pTis carcinoma-in-adenoma lesions and 36 pT3/p Stage II carcinomas of the colon by in-situ hybridization and immunohistochemistry, respectively. RESULTS: 15LOX-1 expression was found in 89% (48 of 54) of adenomas, 43% (nine of 21) of adenomas and 10% (two of 21) of carcinomas in carcinoma-in-adenoma lesions, but not in pT3 carcinomas (P < 0.0001). In contrast, COX-2 production was found in 11% (six of 54) of adenomas, 52% (11 of 21) of adenomas and 71% (15 of 21) of carcinomas in carcinoma-in-adenoma lesions, and 92% (33 of 36) of pT3 carcinomas (P < 0.0001). Concurrence of 15LOX-1 down-regulation and COX-2 up-regulation was found in 6% (three of 54) of adenomas, 33% (seven of 21) of adenomas and 71% (15 of 21) of carcinomas in carcinoma-in-adenoma lesions, and 92% (33 of 36) of pT3 carcinomas (P < 0.0001). CONCLUSIONS: These results suggest that switching of LA metabolism by reversal of the expression of 15LOX-1 and COX-2 is associated with acquisition of malignant potential in colonic neoplasia.

Receptor for advanced glycation end products (RAGE) is important in the prediction of recurrence in human oral squamous cell carcinoma.

AIMS: Receptor for advanced glycation end products (RAGE) has recently been recognized as a cancer-associated protein responsible for cancer progression and metastasis in gastrointestinal cancers. The aim was to examine the role of RAGE in oral squamous cell carcinoma (OSCC). METHODS AND RESULTS: RAGE expression was examined by immunohistochemistry in 74 OSCC patients and evaluated with a grading based on Allred's score. RAGE expression was compared with clinicopathological parameters including clinical stage, invasive depth, nodal metastasis, disease recurrence and disease-free survival. High-grade expression of RAGE (RAGE-H) was observed in 30 (40.5%) of 74 OSCCs. RAGE-H was associated with depth of invasion (P < 0.0001) and local recurrence (P < 0.0001), but not with histological differentiation, clinical stage or nodal metastasis. Disease-free survival in patients with RAGE-H was significantly worse than in those with low-level RAGE expression. Multivariate analysis showed RAGE-H to be an independent prognostic factor for disease-free survival in OSCC patients (P = 0.0022). CONCLUSION: RAGE is a relevant factor in predicting disease recurrence and patients' prognosis in OSCC.

Reg IV is a serum biomarker for gastric cancer patients and predicts response to 5-fluorouracil-based chemotherapy.

Regenerating gene family, member 4 (Reg IV), a secreted protein, is overexpressed in several cancers, including gastric cancer (GC). In the present study, we measured Reg IV levels in sera from patients with GC by enzyme-linked immunosorbent assay. We also examined the effect of forced Reg IV expression on the apoptotic susceptibility to 5-fluorouracil (5-FU). Forced expression of Reg IV inhibited 5-FU-induced apoptosis. Induction of Bcl-2 and dihydropyrimidine dehydrogenase was involved in inhibition of apoptosis. Among 36 GC patients treated with a combination chemotherapy of low-dose 5-FU and cisplatin, all 14 Reg IV-positive patients showed no change or disease progression. The serum Reg IV concentration was similar between healthy individuals (mean+/-s.e., 0.52+/-0.05 ng/ml) and patients with chronic-active gastritis (0.36+/-0.09 ng/ml). However, the serum Reg IV concentration in presurgical GC patients was significantly elevated (1.96+/-0.17 ng/ml), even at stage I. The diagnostic sensitivity of serum Reg IV (36.1%) was superior to that of serum carcinoembryonic antigen (11.5%) or carbohydrate antigen 19-9 (13.1%). These results indicate that expression of Reg IV is a marker for prediction of resistance to 5-FU-based chemotherapy in patients with GC. Serum Reg IV represents a novel biomarker for GC.

Determinants for prediction of malignant potential by metalloproteinase:E-cadherin ratio in prostate core needle biopsy.

According to a good correlation between in situ hybridization-based metalloproteinase-2/9:E-cadherin ratio (MER) and the pathological stage of prostate cancer, we set the cutoff line of MER at 6.0 (MER>6) to distinguish between organ-confined (pT2) and advanced diseases (pT3a-b/N1). In this study, we looked at the factors affecting MER and leading to a misprediction of the pathological stage. We examined MER in 39 paired specimens of prostate core needle biopsy and prostatectomy from the same patient and compared these MERs. In 34 (87%) of 39 cases, the MER of biopsy was correlated with the final pathological stage (pT2 vs. pT3a-b/N1). MER ranges in pT3a-b/N1 cancer were significantly wider than those in pT2 cancer (p < 0.01). The number of MER>6 fields in Gleason score 8-9 cancer was larger than that in Gleason score 7 cancer (p < 0.0001). In 5 cases where there was a failure to distinguish pT2 from pT3a-b/N1, the misdiagnosis was significantly associated with a small number of biopsies (4 or 6 specimens; p = 0.0469), a small amount of tumor tissue in biopsy specimens (less than 5 mm; p = 0.0492), and a wide MER range (more than 5.0; high intratumoral heterogeneity; p = 0.0202). Considering these factors increases the usefulness of preoperative prediction of the final pathological stage by MER in prostate cancer.

Interleukin-15 and transforming growth factor alpha are associated with depletion of tumor-associated macrophages in colon cancer.

We examined the effects of IL-15 and TGF-alpha on the inhibition of macrophage infiltration into colon cancer tissue, and secretion of amphoterin from colon cancer cells. Production of IL-15 and/or TGF-alpha was associated with depletion of tumor-associated macrophages (TAMs) in both Dukes' B and C tumors (P = 0.0324 and 0.0051, respectively). Production of IL-15 and/or TGF-alpha was also associated with amphoterin mRNA expression in colon cancer tissues with TAM depletion in both Dukes' B and C tumors (P = 0.0167 and P = 0.0062, respectively). WiDr human colon cancer cells treated with IL-15 and/or TGF-alpha induced reduction of nucleus-localized amphoterin and an increase in cytosolic and membranous amphoterin. Moreover, IL-15 and/or TGF-alpha treatment increased amphoterin secretion by WiDr cells. Most notably, IL-15 and TGF-alpha treatment induced the increase of cytosol/membrane localization and secretion of amphoterin and the most pronounced effect among the treatments carried out. These results suggested that IL-15/TGF-alpha promotes depletion of TAMs and secretion of amphoterin in colon cancer.

High concentration of deoxycholic acid abrogates in vitro transformation of IEC6 intestinal cells by azoxymethane.

In this study, we designed an in vitro azoxymethane (AOM)-induced carcinogenesis model and analyzed the effect of deoxycholic acid (DCA) on growth, apoptosis, genotoxicity, and transformation of IEC6 intestinal cells. CYP2E1 production was confirmed in IEC6 cells. The growth of IEC6 cells was enhanced by DCA (100 microg/ml). However, IEC6 cells treated with DCA (200 microg/ml) were inhibited and disappeared at 48 hrs after treatment. Apoptotic cells increased 11.2 times by treatment with DCA (200 microg/ml) as compared to cells with no treatment. DNA injury detected by comet assay was found in cells treated with AOM, but not in cells treated with DCA (100 microg/ml) and AOM. The number of colony formation in soft agar increased by AOM treatment. However, the number of foci treated with DCA (100 microg/ml) plus AOM was 69% that of cells treated with AOM alone. Two out of the 6 mice subcutaneously injected with AOM-treated IEC6 cells showed tumorigenesis, whereas IEC6 cells treated with DCA (100 microg/ml) plus AOM or DCA (100 microg/ml) alone did not form any tumor. Reduced protein expression of MLH1, Bcl-2 was detected in IEC6 cells treated with DCA (100 microg/ml). Production of Bax, pJNK, TGF-beta, TGFBRI, TGFBRII, and beta-catenin were higher in IEC6 cells treated with DCA (100 microg/ml) than that in cells with no treatment. These results suggest that high-dose DCA induced apoptosis and inhibited AOM-induced in vitro transformation of IEC6 cells.

Repression of MLH1 and MGMT genes in colon mucosa adjacent to implanted cancer in athymic mouse.

Hyperplastic mucosa adjacent to colon cancer, being a reactive change, accelerates cancer progression and its metastasis through expression of angiogenic factors. We investigated promoter methylation in hyperplastic mucosa adjacent to orthotopic KM12SM colon cancer in mice. In the hyperplastic mucosa adjacent to KM12SM tumors in the cecum of athymic mice, reductions in the levels of the mutL homologue 1 (MLH1) and O6-methylguanine-DNA methyltransferase (MGMT) proteins were detected by immunohistochemistry and immunoblotting. To examine the effects of growth factors and cytokines on promoter methylation and repressed expression of the MLH1 and MGMT genes, a rat intestinal epithelial cell line, IEC6, was treated with epidermal growth factor (EGF) and interleukin (IL)-15 for 35 days. Protein levels of MLH1 and MGMT were reduced in EGF- and IL-15-treated IEC6 cells. A methylation-sensitive restriction enzyme assay revealed that CpG methylation was present in the promoter regions of the MLH1 and MGMT genes in DNAs extracted from hyperplastic mucosa adjacent to KM12SM tumors. These findings suggest that promoter CpG methylation affects expression of MLH1 MGMT genes in hyperplastic mucosa adjacent to colon cancer.

Suppressive effects of a selective cyclooxygenase-2 inhibitor, etodolac, on 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

The present study was conducted to examine effects of a clinically available selective cyclooxygenase (COX)-2 inhibitor, etodolac, on the development of rat tongue squamous cell carcinomas (SCCs) induced by 4-nitroquinoline 1-oxide (4-NQO), and on the immunohistochemically demonstrable expression of COX-2. Fischer 344 rats, 6 weeks old at the commencement, were administered 4-NQO at the doses of 20-30 ppm in their drinking water for 12 weeks. Then, etodolac was supplemented into the diet at doses of 150 and 300ppm for 16 weeks. Rats were sacrificed at 28 weeks and tongue lesions were histologically examined. The incidence and the multiplicity of SCCs induced by 4-NQO were dose-dependently reduced by etodolac, with significance at the highest dose of 300 ppm. Etodolac did not significantly affect the immunohistochemical expression of COX-2 in the lesions which did develop. These results indicate that etodolac can inhibit the development of rat tongue SCCs, probably by inhibiting COX-2 activity rather than its expression. Thus, etodolac may be a promising candidate chemopreventive agent for individuals at high risk of oral cancer.

Acetylated histone H4 is reduced in human gastric adenomas and carcinomas.

Acetylation of core histones is closely linked to transcriptional activation of various genes. The acetylation levels of nucleosomal histones can be modified through a balance of histone acetyltransferases and deacetylases. To elucidate the role of histone acetylation in human gastric carcinogenesis, we studied the status of histone H4 acetylation in gastric carcinoma tissues and corresponding non-neoplastic mucosa. The status of histone acetylation was assessed by examining the expression of acetylated histone H4 through Western blotting and immunohistochemistry using an anti-acetylated histone H4 antibody. The levels of acetylated histone H4 expression were obviously reduced in 72% (13/18) of gastric carcinomas in comparison with non-neoplastic mucosa by Western blotting. In immunohistochemistry, acetylated histone H4 was clearly detected in the nuclei of both non-neoplastic epithelial and stromal cells, whereas the levels of acetylated histone H4 were heterogeneous or reduced in 66% (38/57) of gastric carcinomas and 46% (6/13) of gastric adenomas. Reduced expression of acetylated histone H4 was also observed in some areas of intestinal metaplasia adjacent to carcinomas. Reduction in the expression of acetylated histone H4 was significantly correlated with advanced stage, depth of tumor invasion and lymph node metastasis. These results suggest that low levels of histone acetylation may be closely associated with the development and progression of gastric carcinomas, possibly through alteration of gene expression.

Heparan sulfate enhances invasion by human colon carcinoma cell lines through expression of CD44 variant exon 3.

CD44 variant exon (CD44v) 3 is a heparan sulfate-binding isoform of CD44. The role of CD44v3 in invasion and metastasis associated with heparan sulfate in colon cancer cell lines and cases of colon cancer was examined. Expression of CD44v3 mRNA and protein was observed in five of six human colorectal cancer cell lines. Colo320 and WiDr cells expressed CD44v3 at high levels. Heparan sulfate treatment increased the invasive activity of Colo320 and WiDr cells to rates 14.3 and 12.6 times higher, respectively, than that of untreated cells. However, heparan sulfate treatment did not affect cell growth. Repression of CD44v3 protein production by antisense S-oligodeoxynucleotide treatment reduced the binding affinities and capacities for heparan sulfate by Colo320 and WiDr cells in comparison with that of control cells, and it also reduced the invasiveness of both cell lines to one-fifth that of control cells. In heparan sulfate-treated Colo320 cells, the levels of CD44v3 protein in the Triton X-100-insoluble fraction and moesin-precipitated fraction were increased, suggesting that heparan sulfate treatment facilitates association of CD44 molecules with the cytoskeleton. Immunohistochemical analysis showed CD44v3 to be expressed in 21 of 37 (57%) colorectal cancer cases. Positive CD44v3 expression was associated with more advanced pathological stage and poorer prognosis than negative CD44v3 expression. These data support a role for CD44v3 in invasion and metastasis by colorectal carcinoma cells.

Interleukin-15 expression is associated with malignant potential in colon cancer cells.

Interleukin 15 (IL-15 mRNA expression was detected in human colorectal cancer cells (Colo320, WiDr, TCO and DLD1) by the reverse transcriptase-polymerase chain reaction (RT-PCR). Only Colo320 and WiDr cells secreted IL-15 culture medium. With IL-15 treatment, all cell lines grew at a rate of 120-180% of that of nontreated cells. A binding assay with (125)I-labeled IL-15 showed binding activity to IL-15 in Colo320 (K(d): 0.098 nM) cells. IL-15 also reversed the growth inhibition caused by serum starvation in Colo320 cells. IL-15-induced cell growth in regular and serum-free media was abrogated by anti-IL-15 antibody treatment in Colo320 cells. Moreover, IL-15 treatment reduced doxorubicin-induced cytostasis and cytolysis in Colo320 cells by 50%. The invasion capacity of IL-15-treated Colo320 cells was 5.3 times that of untreated cells. Immunoblotting showed that IL-15-treated Colo320 cells exhibited downregulation of p21Waf1 and Bax, and upregulation of Bcl-2, phospho-AKT, MMP9/MMP2, and VEGF. Finally, immunostaining of human colon cancer revealed that 33 (70%) of 47 Dukes' C cases showed IL-15 expression in cancer cells, whereas only 16% of Dukes' B cases did (p < 0.0001). IL-15 may play important roles in cell proliferation, invasion, and metastasis of human colorectal cancer.

Expression of Bub1 gene correlates with tumor proliferating activity in human gastric carcinomas.

Bub1 plays an important role at the spindle assembly checkpoint to prevent cell cycle progression following spindle damage. We examined the expression of human Bub1 mRNA in 20 gastric carcinoma tissues and corresponding nonneoplastic mucosas by reverse transcriptase-polymerase chain reaction and analyzed the relation with proliferative activity monitored by the expression of proliferating cell nuclear antigen (PCNA) on Western blotting as well as Ki-67 labeling index by immunohistochemistry. Increased expression of Bub1 mRNA was detected in 8 (40%) of the gastric carcinomas in comparison with their nonneoplastic counterparts, while 4 (20%) expressed Bub1 at lower levels. The expression of Bub1 mRNA was confirmed by in situ hybridization. The expression levels of Bub1 mRNA were well correlated with the levels of PCNA protein in 16 (80%) gastric carcinoma cases. The examination of Ki-67 labeling indices proved the close correlation between the expression levels of Bub1 and proliferating activity. These findings suggest that mRNA expression of human Bub1 gene is closely associated with the tumor-proliferating activity. Since genetic alterations of human Bub1 rarely occur in gastrointestinal cancers, the functional machinery of Bub1 to prevent cell cycle progression into anaphase might be well preserved in gastric carcinomas even with high proliferative activity.

Interferon-alpha prevents selection of doxorubicin-resistant undifferentiated-androgen-insensitive metastatic human prostate cancer cells.

BACKGROUND: We determined whether treatment of metastatic prostate cancer cells with doxorubicin (DOX) and interferon-alpha (IFN-alpha) prevented the emergence of highly undifferentiated tumor cells. METHODS: The state of cell differentiation was determined by analysis of prostate-specific antigen (PSA), E-cadherin, keratin, and vimentin. RESULTS: Human prostate cancer LNCaP-LN3 cells growing in culture as multicell spheroids expressed higher levels of E-cadherin and E-cadherin-associated beta-catenin than LNCaP-LN3 cells growing as monolayers. Treatment of cells with DOX downregulated PSA, E-cadherin, and keratin, and upregulated expression of vimentin and vascular endothelial growth factor (VEGF) mRNA. While treatment of cells with IFN-alpha did not alter gene expression, the addition of IFN-alpha to cultures treated with DOX produced synergistic toxicity and abrogated the changes in gene expression observed in cells treated with DOX alone. CONCLUSIONS: Treatment with IFN-alpha and DOX should be further explored as a therapeutic strategy for androgen-insensitive prostate cancer.