Indole-3-carbinol inhibits the proliferation of colorectal carcinoma LoVo cells through activation of the apoptotic signaling pathway.

Indole-3-carbinol (I3C) is a phytochemical that exhibits growth-inhibitory activity against various cancer cells. However, there are limited studies on the effects of I3C on colon cancer cells. In this study, the growth-inhibitory activity of I3C against the human colorectal carcinoma cell line (LoVo) was examined. The results of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, colony formation, and cell counting assays revealed that I3C suppressed the proliferation of LoVo cells. Microscopy and wound-healing analyses revealed that I3C affected the morphology and inhibited the migration of LoVo cells, respectively. I3C induced apoptosis and DNA fragmentation as evidenced by the results of fluorescein isothiocyanate-conjugated annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay, respectively. Additionally, I3C arrested the cell cycle at the G0/G1 phase and enhanced the reactive oxygen species levels. Western blotting analysis revealed that treatment with I3C resulted in the activation of apoptotic proteins, such as poly(ADP-ribose) polymerase, caspase-3, caspase-7, caspase-9, Bax, Bim, and p53 in LoVo cells. These results indicate that I3C induces apoptosis in LoVo cells by upregulating p53, leading to the activation of Bax and caspases. Taken together, I3C exerts cytotoxic effects on LoVo cells by activating apoptosis.

Cytoprotective effects of taxifolin against cadmium-induced apoptosis in human keratinocytes.

Cadmium (Cd) is a heavy metal widely used in industry, and the skin is an important target of this metal. Taxifolin (Tax), a natural source of bioflavonoids found in various conifers, exerts multiple biologic effects on skin cells. However, the mechanisms by which Tax protects keratinocytes against Cd are currently unclear. We investigated the cytoprotective effects of Tax against Cd-induced apoptosis in the human HaCaT keratinocyte. The water-soluble tetrazolium salt (WST-1) assay and Annexin V/propidium iodide double-staining assay results showed that Cd-induced cell death was lower in cells treated with Tax (0-100 µM) than in cells treated with Cd alone. Additionally, a reduction of Cd-induced DNA fragmentation by Tax was shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay. The levels of reactive oxygen species were also lower in Cd/Tax-treated cells than in Cd-treated cells. We employed a two-dimensional electrophoresis-based proteomic analysis to identify treatment-related alterations in protein expression. Tax downregulated cathepsin B and D and upregulated hsp27, cyclophilin A, and peroxiredowin-1. Western blotting confirmed the downregulation of cathepsin B and D and the upregulation of hsp27. The cytoprotective effects of Tax against Cd-induced apoptosis were also characterized by the changes in the activity of caspase 3, -7, poly ADP-ribose polymerase, the cellular proliferation-related ERK1/2, and AKT. Furthermore, the levels of cell cycle-related proteins, such as SP1 and p21, decreased, whereas p53 level increased. We concluded that Tax reduced Cd cytotoxicity and Cd-induced apoptosis by inhibiting the apoptotic pathway.

Anticarcinogenic effect of indole-3-carbinol (I3C) on human hepatocellular carcinoma SNU449 cells.

Many cruciferous vegetables, including cabbage, contain indole-3-carbinol (I3C), which is a known anticarcinogen. However, the anticarcinogenic effects of I3C on liver cancer have not been investigated. Therefore, this study was conducted to evaluate the anticarcinogenic effects of I3C in human hepatocellular carcinoma (HCC) SNU449 cells. The results of MTT and WST-1 assays indicated that treatment of SNU449 cells with I3C decreased viability in dose- and time-dependent manners, while colony formation assays indicated that I3C also inhibited proliferation of SNU449 cells. Moreover, fluorescence-activated cell sorter analysis showed that I3C induced apoptosis in SNU449 cells in dose- and time-dependent manners. Furthermore, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling revealed that I3C induced DNA fragmentation in SNU449 cells in a time-dependent manner, while Western blotting showed that apoptotic proteins such as p53, cleaved PARP, caspase-3, and caspase-7 were activated in SNU449 cells following treatment with I3C. Finally, reactive oxygen species-related protein peroxiredoxin-1 and thioredoxin-1 expression decreased in I3C-treated SNU449 cells. The aim of our study is to investigate the unknown mechanisms responsible for the apoptotic effects of I3C on human HCC SNU449 cells, and the results suggest that I3C may be useful for the prevention and treatment of liver cancer.

Long-term outcomes of locally or radically resected T1 colorectal cancer.

AIM: Little is known about the long-term outcome of T1 colorectal cancer (CRC) following curative resection. The present study addressed the long-term outcome of locally or radically resected T1 CRCs. METHOD: A total of 430 patients with T1 CRC who underwent local or radical resection were considered. Unfavourable histological factors were defined as positive resection margin, deep submucosal invasion, vascular invasion, Grade 3 and budding. The patients were classified as low-risk (unfavourable histological factor negative, n = 65) or high-risk (unfavourable histological factor positive, n = 365). RESULTS: Over a median follow-up of 78.4 months, disease recurred in 16 (3.7%) patients in the high-risk group, and no recurrence in the low-risk group. Resection type and vascular invasion were significantly associated with recurrence. In the vascular invasion (+) high-risk group, both 5-year disease-free survival rate and 5-year overall survival rate were significantly associated with resection type (radical 94.6%, local 43.8%, P < 0.001, and radical 99.1%, local 66.7%, P < 0.001). In the vascular invasion (-) high-risk group, 5-year disease-free survival rate was also significantly associated with resection type (radical 98.9%, local 84.7%, P = 0.001). However, 5-year overall survival rate was not associated with resection type (radical 98.9%, local 95.2%, P = 0.816). CONCLUSION: Local resection may be effective and oncologically safe in low-risk T1 CRC. Although additional surgery should be recommended for the locally resected high-risk T1 CRC cases, intensive surveillance without additional surgery and timely salvage operation may offer another treatment option, if vascular invasion is negative.

Effects of human epidermal growth factor gene-transfected mesenchymal stem cells on fibroblast migration and proliferation.

OBJECTIVES: We were interested in determining whether epidermal growth factor gene-transfected mesenchymal stem cells (EGF-MSC) would accelerate fibroblast migration and proliferation. MATERIALS AND METHODS: Fibroblasts were cultured in serum-free conditioned media from EGF-MSC; RT-PCR was performed to detect expression of EGF gene in EGF-MSCs. EGF protein levels in cell culture supernatants from EGF-MSC were assayed by ELISA and proliferation of EGF-MSC-treated fibroblasts was performed using MTT assay. Effects of EGF-MSC on fibroblast migration were evaluated using scratch wound and transmigration assays. Cell adhesion molecules, cell dynamics molecules and phospho-(Ser) kinase substrate expressions of EGF-MSC-treated fibroblasts were evaluated by western blotting. RESULTS: EGF gene expression increased in EGF-MSCs and viability of EGF-MSC-treated fibroblasts was elevated. EGF-MSC-treated fibroblasts showed increased migration compared to controls. Expressions of cell adhesion molecules (ß-catenin, N-cadherin), cell dynamics molecules (cofilin, ezrin) and phospho-(Ser) kinase substrates (phospho-MAPK/CDK substrate, phospho-Arg-(Ser)-X-Tyr/Phe-X-pSer motif) increased in EGF-MSC-treated fibroblasts. These results imply that EGF-MSCs contributed to enhancing the wound healing process by increased cell adhesion, dynamic effects, fibroblast migration, and proliferation. CONCLUSIONS: This study indicates that EGF-MSCs had a positive influence on fibroblast migration and proliferation and EGF-MSC may provide a useful strategy for wound healing.

Induction of angiogenesis by expression of soluble type II transforming growth factor-beta receptor in mouse hepatoma.

The biological effect of transforming growth factor-beta (TGF-beta) is cell type-specific and complex. The precise role of TGF-beta is not clear in vivo. To elucidate the regulation mechanism of endogenous TGF-beta on hepatoma progression, we modified the MH129F mouse hepatoma cell with a retroviral vector encoding the extracellular region of type II TGF-beta receptor (TRII). Soluble TRII (TRIIs) blocked TGF-beta binding to TRII on the membrane of hepatoma cells. Growth of MH129F cells was inhibited by TGF-beta1 treatment; however, soluble TRII-overexpressing cells (MH129F/TRIIs) did not show any change in proliferation after TGF-beta1 treatment. MH129F/TRIIs cells also increased vascular endothelial growth factor (VEGF) expression, endothelial cell migration, and tube formation. Implantation of MH129F/TRIIs cells into C3H/He mice showed the significantly enhanced tumor formation. According to Western blot and protein kinase C assay, the expression of VEGF, KDR/flk-1 receptor, and endothelial nitric-oxide synthase was enhanced, and the phosphorylation activity of protein kinase C was increased up to 3.7-fold in MH129F/TRIIs tumors. Finally, a PECAM-1-stained intratumoral vessel was shown to be 4.2-fold higher in the MH129F/TRIIs tumor. These results indicate that VEGF expression is up-regulated by a blockade of endogenous TGF-beta signaling in TGF-beta-sensitive hepatoma cells and then stimulates angiogenesis and tumorigenicity. Therefore, we suggest that endogenous TGF-beta is a major regulator of the VEGF/flk-1-mediated angiogenesis pathway in hepatoma progression.

Enhancement of natural killer cell-mediated cytotoxicity by coexpression of GM-CSF/B70 in hepatoma.

On investigating the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and costimulatory molecule, B70, in antitumor immunity, we have found important effects of GM-CSF/B70 coexpression in the interaction with natural killer (NK) cells. We used the pLSN vector system to contain the neomycin-resistant gene and LTR promoter. The pLSNGM-CSF, pLSNB70 and pLSNB70/GM-CSF, pLSN vectors each containing GM-CSF, B70, and B70/GM-CSF cDNA, respectively, were constructed. They were transfected into human hepatocellular carcinoma cell (SK-HEP1), and stable cells (SK-pLSN, SK-GM, SK-B70 and SK-BG) were selected after neomycin treatment. According to enzyme-linked immunosorbent analysis and FACS, we showed that expression of GM-CSF was increased up to 23-fold in SK-GM and SK-BG cells, and also expression of B70 was induced at least 76-97% in SK-B70 and SK-BG cells. Expression of B70 was remarkably increased by autocrine effect of GM-CSF in SK-BG cells. Primary cytolytic ability of GM-CSF and B70 significantly increased almost 4-fold (effector/target ratio, 100:1) in SK-BG cells. In in vivo studies, SK-BG cells showed much less subcutaneous tumor formation in nude mice accompanying increased NK cell proliferation and cytotoxicity. Therefore, these results suggest that combining expression of GM-CSF and B70 may enhance NK-mediated cytotoxicity, and then induce the antitumor immunity in hepatoma transplanted into nude mice.

Activated ras oncogene collaborates with HBx gene of hepatitis B virus to transform cells by suppressing HBx-mediated apoptosis.

The hepatitis B virus HBx protein is a promiscuous transactivator implicated in the development of hepatocellular carcinoma. The ectopic expression of HBx fails to transform both primary and immortalized rodent cells, but rather induces apoptosis. Furthermore, most transgenic mice harboring HBx do not develop liver tumors. Thus, it remains unclear whether and how HBx contributes to oncogenesis. Here, we show that HBx collaborates with activated H-ras to transform immortalized rodent cells. Indeed, REF52 cells transfected by both HBx and activated H-ras were morphologically transformed and were able to grow in soft agar. Remarkably, nude mice injected with REF52 cells transfected by both HBx and activated H-ras developed tumors, whereas the mice injected with REF52 cells transfected by either gene alone did not. Thus, we concluded that HBx could contribute to neoplastic transformation of cells in collaboration with other oncogenes, such as H-ras, that renders cells to overcome the HBx-mediated apoptosis. Further, we found that HBx mediated apoptosis was suppressed by activated H-ras through activation of the phosphatidylinositol-3 kinase and Akt pathway. Data presented here firmly established the oncogenic potential of HBx during multistage carcinogenesis. Oncogene (2001) 20, 16 - 23.

Studies of cocktail therapy with multiple cytokines for neoplasia or infectious disease of the dog I. cDNA cloning of canine IL-3 and IL-6.

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.

Cloning of canine GM-CSF and SCF genes.

Cytokines have pleiotropic regulatory effects on hematopoietic cells and many other cell types that participate in host defence and repair processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages and regulates the biological functions expressed by mature cells of these lineages. Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. In order to determine the complementary DNA (cDNA) of canine GM-CSF and canine SCF, cDNA clones were generated from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) and bone marrow cells by reverse transcription PCR amplification. The canine GM-CSF cDNA obtained in this study contains an open reading frame encoding 144 amino acid residues and has 53-75% homology with those of human, cat, sheep, pig, cow and mouse, Canine SCF cDNA consist of an open reading frame encoding 274 amino acid residues and shares 81-92% homology with those of human, cat, pig, cow and mouse.

The growth inhibition of hepatoma by gene transfer of antisense vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and tumor growth in solid tumors. Therefore, to induce tumor regression, antiangiogenic agents to block VEGF need to be administered repeatedly. METHOD: We constructed the recombinant mammalian expression vector bearing an antisense-VEGF cDNA, pZeoVEGFa. We examined the effect of pZeoVEGFa on the growth of SK-HEP1 hepatoma cells, bovine capillary endothelial (BCE) cells, and tubule formation of BCE cells in fibrin gel. To evaluate the function of pZeoVEGFa in vivo, we implanted SK-HEP1 hepatoma cells subcutaneously into nude mice. RESULTS: In SK-HEP1 hepatoma cells, we showed that the synthesis of VEGF protein was suppressed by the stable and transient transfection of pZeoVEGFa. pZeoVEGFa inhibited the proliferation of BCE cells and significantly suppressed tubule formation of BCE cells. pZeoVEGFa inhibited a morphological change from a round shape to an elongated spindle shape in fibrin gel. When pZeoVEGFa was injected peritumorally by liposomes, tumor growth was inhibited. CONCLUSION: Endothelial cell proliferation, tubule formation and tumor growth may be diminished by down-regulation of endogenous VEGF expression in tumor cells or tissue. These findings indicate that the efficient down-regulation of the VEGF produced by tumor cells using antisense strategies has an antitumor effect. We suggest that VEGF-targeted antiangiogenic gene therapy could be an effective strategy to treat VEGF-producing tumors.

Immunofluorescent localization of constitutive and inducible prostaglandin H synthase in ovine astroglia.

We have used immunofluorescent techniques to examine the distribution of prostaglandin H synthase (PGHS) in ovine astrocyte-enriched secondary cultures and in mixed cortical cells in primary culture. A battery of monoclonal and polyclonal antibodies specific for the constitutive (PGHS-1) or inducible (PGHS-2) forms of the enzyme were used to examine the cells in culture. Varying levels of PGHS-1 and PGHS-2-specific immunofluorescence were seen in astrocytes as well as in other cells. The fluorescent pattern and localization seen with antisera to both PGHS-1 and PGHS-2 were similar but were not identical. Both immunoreactive species were confined to nuclear and perinuclear regions of the cell, with no immunoreactivity evident in plasmalemma. In addition, PGHS-2-specific fluorescence was concentrated often as a homogeneous ring around the nucleus in heavily stained astrocytes. Mixed cortical glia/fibroblasts in primary culture were double labeled with antibodies to glial fibrillary acidic protein (GFAP) and to PGHS-2. GFAP and PGHS-2 were colocalized in clusters of astrocytes, but PGHS-2 was evident in GFAP- cells as well. Cells treated with the mitogenic agent phorbol dibutyrate displayed more PGHS-2+ immunofluorescence compared to either vehicle control or cells pretreated with dexamethasone. We conclude that astrocytes cultured in serum express both constitutive and inducible forms of PGHS and that PGHS-2 is induced by mitogens in this cell type.

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia.

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F2a (PGF2 alpha) levels in media were determined at 2-24 hours after exposure to PDB. PDB increased production of PGF2 alpha at 10(-8)M and 10(-6)M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10(-4)M) with PDB (10(-6)M) inhibited PDB-induced PGF2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.

Role of protein synthesis in interleukin 1 alpha-induced prostaglandin production in ovine astroglia.

Experiments were done to determine whether interleukin 1 alpha (IL-1 alpha) induces prostaglandin (PG) synthesis in cultured astroglia and to examine the role of enhanced cyclooxygenase 2 (COX 2) synthesis in these responses. Immunologically identified fetal ovine astrocytes were exposed to IL-1 alpha or to media alone. Application of 10 ng/ml IL-1 alpha increased the production of PGF2 alpha for 2-24 h. Coincubation of IL-1 alpha with actinomycin D (1 microgram/ml) or cycloheximide (10 micrograms/ml) completely blocked the increase in PGF2 alpha. Immunoblot analyses revealed that the synthesis of COX2 was not increased by IL-1 alpha at 4 h. However, with incubation of IL-1 alpha for 8-12 h, COX2 levels were increased. These results indicate that longer periods than 4 h are necessary to increase COX2 protein, suggesting that proteins other than COX2 are involved in early IL-1 alpha-induced PG synthesis.

Protein kinases and prostaglandin production in ovine astroglia.

We examined the effects of interleukin-1 alpha (IL-1 alpha) and involvement of protein kinases on prostaglandin production in cultured ovine astroglia. Ovine astroglia were exposed to media alone, or 10 ng/mL IL-1 alpha and prostaglandin F2 alpha (PGF2 alpha) levels were analyzed using enzyme immunoassay. Application of IL-1 alpha augmented the production of PGF2 alpha at 4 h. Coapplication of H-7 (10-1000 microM) and staurosporine (0.1-10 microM), inhibitors of protein kinase C (PKC), blocked IL-1 alpha-induced PGF2 alpha production. IL-1 alpha increased cyclooxygenase (COX) activity while coapplication of staurosporine prevented an increase, implying that COX activity was dependent upon PKC activation. In contrast, forskolin, sodium nitroprusside, and cyclic nucleotide analogs alone did not affect prostaglandin production significantly, excluding the involvement of cAMP/cGMP-dependent protein kinases. Coapplication of quinacrine (10 microM) and bromophenacyl bromide (100 microM), inhibitors of phospholipase A2 (PLA2), prevented the IL-1 alpha-induced increases in PGF2 alpha production. Lastly, IL-1 alpha increased labeled arachidonic acid (AA) release whereas coaddition of quinacrine (10 microM) attenuated increased AA release. Therefore, we propose that IL-1 alpha enhances prostaglandin production by ovine astroglia via steps involving activation of PKC and increased activity of COX and PLA2.

Rapid induction of prostaglandin synthesis in piglet astroglial cells by interleukin 1 alpha.

We examined effects of interleukin 1 alpha (IL-1 alpha) on prostaglandin production in piglet cultured astroglia. Immuno- and morphologically identified polymorphic and process-bearing astrocytes were collected from cerebral cortex and white matter from piglets (1-3 days of age). Levels of prostaglandins were determined using enzyme immunoassay. Baseline levels for prostaglandin (PG) F2 alpha were 631 +/- 332 pg/ml and increased to 1417 +/- 353 pg/ml 20 min after addition of 11 micrograms/ml IL-1 alpha (p < 0.05) and to 2280 +/- 391 pg/ml 20 min after addition of 22 micrograms/ml IL-1 alpha (p < 0.05) (n = 7). Only small amounts of 6-keto-PGF 1 alpha or PGE2 were detected in medium under baseline conditions or in the presence of IL-1 alpha. Medium alone did not change PGF2 alpha levels (n = 3). Coapplication of cycloheximide (10(-3) M) blocked the increase in PGF2 alpha (n = 3). We conclude that IL-1 alpha causes a rapid increase in prostaglandin production by astroglia, and this process involves a step requiring continued or increased protein synthesis.