Showing papers in "Molecular Carcinogenesis in 2004"

Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells.

Abstract: There is an emerging evidence that plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) may have potential as a chemotherapeutic agent. However, the growth inhibitory mechanisms of plumbagin have remained unexplored. The aim of the study was to determine whether plumbagin-induced cell death in human cervical cancer cell line, ME-180, exhibited biochemical characteristics of apoptosis and to check whether N-acetyl-l-cysteine (NAC), which is a free radical scavenger, can reverse the cytotoxic effects of plumbagin. It can be concluded from the results that plumbagin inhibits the growth of ME-180 cells in a concentration and time-dependent manner. The cytotoxic effect of plumbagin induced cell death is through the generation of reactive oxygen species (ROS) and subsequent induction of apoptosis as demonstrated by the present data. Treatment of cells with plumbagin caused loss of mitochondrial membrane potential (DeltaPsi(m)), and morphological changes characteristic of apoptosis, such as the translocation of phosphatidyl serine, nuclear condensation, and DNA fragmentation. Moreover, plumbagin-induced apoptosis involved release of mitochondrial cytochrome c and apoptosis inducing factor (AIF), thus activation of caspase-dependent and -independent pathways, as shown by the plumbagin-mediated activation of caspase-3 and -9. Our results also show that pretreatment of ME-180 cells with NAC blocks plumbagin-induced loss of DeltaPsi(m) and subsequent release of cytochrome c, AIF, and caspase-9 and -3 activation, thus inhibiting the apoptotic ability of plumbagin.

Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2.

Abstract: Cancer metastasis, involving multiple processes and various cytophysiological changes, is a primary cause of cancer death and may complicate the clinical management, even lead to death Silibinin is a flavonoid antioxidant and wildly used for its antihepatotoxic properties and recent studies have revealed pleiotropic anticancer and antiproliferative capabilities of silibinin In this study, we first observed that silibinin exerted a dose- and time-dependent inhibitory effect on the invasion and motility, but hardly on the adhesion, of highly metastatic A549 cells in the absence of cytotoxicity To look at the precise involvement of silibinin in cancer metastasis, A549 cells were treated with silibinin at various concentrations, up to 100 microM, for a defined period and then subjected to gelatin zymography, casein zymography and Western blot to investigate the impacts of silibinin on metalloproteinase-2 (MMP-2), urokinase plasminogen activator (u-PA), and tissue inhibitor of metalloproteinase-2 (TIMP-2), respectively The results showed that a silibinin treatment may decrease the expressions of MMP-2 and u-PA in a concentration- and time-dependent manner and enhance the expression of TIMP-2 Further analysis with semi-quantitative RT-PCR showed that silibinin may regulate the expressions of MMP-2 and u-PA on the transcriptional level while on the translational or post-translational level for TIMP-2

Mammary ECM composition and function are altered by reproductive state

Abstract: To address whether reproductive state alters mammary gland extracellular matrix (ECM) composition and function, ECM was isolated from nulliparous, pregnant, lactating, involuting, and regressed rat mammary glands. The ECM composition of fibronectin, tenascin, laminin, clusterin, and MMPs was found to vary dramatically with reproductive state. In 3-dimensional (3-D) culture, we identified novel effects of these endogenous mammary matrices on mammary epithelial cells. Specifically we found that (1) matrix isolated from nulliparous animals promoted the formation of epithelial ducts with bifurcation, (2) matrix isolated from mid-involuting mammary glands induced cell death, (3) matrix isolated from late-stage involuting glands restored glandular development, while (4) matrix isolated from parous animals restricted glandular morphogenesis. Our data were consistent with mammary gland ECM facilitating epithelial cell proliferation, differentiation, death, and glandular reorganization that occur during the pregnancy and involution cycle. Further, we show that the parous gland has persistent changes in ECM function. Cumulatively, our data demonstrated that the microenvironment of the normal adult mammary gland is highly plastic, which has important implications for mammary tumor cell progression and dormancy. These data also raised the possibility of targeting mammary matrix production with preventive or therapeutic interventions.

Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells.

Abstract: Development of effective agents for treatment of hormone-refractory prostate cancer has become a national medical priority. We have reported recently that apigenin (4′,5,7-trihydroxyflavone), found in many common fruits and vegetables, has shown remarkable effects in inhibiting cell growth and inducing apoptosis in many human prostate carcinoma cells. Here we demonstrate the molecular mechanism of inhibitory action of apigenin on androgen-refractory human prostate carcinoma DU145 cells that have mutations in the tumor suppressor gene p53 and pRb. Treatment of cells with apigenin resulted in a dose- and time-dependent inhibition of growth, colony formation, and G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk)2, 4, and 6, with concomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. The induction of WAF1/p21 and its growth inhibitory effects by apigenin appears to be independent of p53 and pRb status of these cells. Apigenin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in a significant increase in cleaved fragments of caspase-9, -3, and poly(ADP-ribose) polymerase (PARP). Further, apigenin treatment resulted in downmodulation of the constitutive expression of nuclear factor-kappaB (NF-κB)/p65 and NF-κB/p50 in the nuclear fraction that correlated with an increase in the expression of IkappaB-alpha (IκBα) in the cytosol. Taken together, we concluded that molecular mechanisms during apigenin-mediated growth inhibition and induction of apoptosis in DU145 cells was due to (1) modulation in cell-cycle machinery, (2) disruption of mitochondrial function, and (3) NF-κB inhibition. © 2004 Wiley-Liss, Inc.

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol

Abstract: Garcinol is a polyisoprenylated benzophenone derivative of Garcinia indica fruit rind and other species. Recent studies have demonstrated that garcinol exhibited antioxidative effects in vitro. In this study, we found that garcinol inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-activated macrophages. Western blot analyzes and gel-shift assays revealed that garcinol strongly blocks the activation of eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B)-induced by LPS. Moreover, transient transfection experiments showed that garcinol inhibited the NF-kappa B-dependent transcriptional activity. Based on these data, we demonstrated that inhibition of LPS-induced NF-kappa B activation occurred through suppressing the phosphorylation of I kappa B alpha and p38 mitogen-activated kinase (MAPK). Garcinol also lowers the LPS-induced increase of intracellular reactive oxygen species (ROS), which contributes to the activation of NF-kappa B. The NF-kappa B signaling pathway leads to inflammatory reaction and our results suggest that garcinol suppresses the expression of iNOS in this pathway.

Imbalance of antioxidant enzymes in tumor cells and inhibition of proliferation and malignant features by scavenging hydrogen peroxide.

Abstract: The aim of this study was to evaluate the endogenous alterations of the antioxidant enzymes in tumor cells and to specifically compensate the resulting changes in the levels of reactive oxygen species (ROS) to control the malignant growth. We determined and compared the activities of antioxidant enzymes and the levels of superoxide anion (O2*-) and hydrogen peroxide (H2O2) in tumor cell lines with different degrees of malignancy, paired with regard to their origin (PB/CH72T4, PDV/PDVC57, and HBL-100/MCF-7). An increase in superoxide dismutase activity and a decrease in the activities of H2O2-detoxifying enzymes, as a function of malignancy, coupled with a rise in H2O2 and a decrease in O2*- were demonstrated. Treatment of cells with exogenous catalase showed a dose-dependent inhibition of proliferation. This inhibition was also demonstrated in several cell lines of different tissue origin and species, suggesting a general role of H2O2 in cell proliferation. Moreover, stable expression of human catalase in MCF-7 cells inhibited proliferation and also reverted malignant features. We conclude that H2O2 played a crucial and general role in the regulation of proliferation and that an endogenous imbalance in antioxidant enzymes could be a relevant event in the carcinogenesis process.

Pharmacological inhibition of fatty acid synthase (FAS): a novel therapeutic approach for breast cancer chemoprevention through its ability to suppress Her-2/neu (erbB-2) oncogene-induced malignant transformation.

Abstract: We designed our experiments to evaluate whether fatty acid synthase (FAS), a lipogenic enzyme linked to tumor virulence in population studies of human cancer, is necessary for the malignant transformation induced by Her-2/neu (erbB-2) oncogene, which is overexpressed not only in invasive breast cancer but also in premalignant atypical duct proliferations and in ductal carcinoma in situ of the breast. To avoid the genetic complexities associated with established breast cancer cell lines, we employed NIH-3T3 mouse fibroblasts engineered to overexpress human Her-2/neu coding sequence. NIH-3T3/Her-2 cells demonstrated a significant upregulation of FAS protein expression, which was dependent on the upstream activation of mitogen-activated protein kinase and phosphatidylinositol 3′-kinase/AKT pathways. Remarkably, pharmacological FAS blockade using the mycotoxin cerulenin or the novel small compound C75 completely suppressed the state of Her-2/neu-induced malignant transformation by inhibiting the ability of NIH-3T3/Her-2 cells to grow under either anchorage-independent (i.e., to form colonies in soft agar) or low-serum monolayer conditions. Moreover, NIH-3T3/Her-2 fibroblasts were up to three times more sensitive to chemical FAS inhibitors relative to untransformed controls as determined by MTT-based cell viability assays. In addition, pharmacological FAS blockade preferentially induced apoptotic cell death of NIH-3T3/Her-2 fibroblasts, as determined by an ELISA for histone-associated DNA fragments and by the terminal deoxynucleotidyltransferase (TdT)-mediated nick end labeling assay (TUNEL). Interestingly, the degree of Her-2/neu oncogene expression in a panel of breast cancer cell lines was predictive of sensitivity to chemical FAS inhibitors-induced cytotoxicity, while low-FAS expressing and chemical FAS inhibitors-resistant MDA-MB-231 breast cancer cells became hypersensitive to FAS blockade when they were engineered to overexpress Her-2/neu. Our observations strongly suggest that inhibition of FAS activity may provide a new molecular avenue for chemotherapeutic prevention and/or treatment of Her-2/neu-related breast carcinomas. © 2004 Wiley-Liss, Inc.

Crossregulation of NF‐κB by the APC/GSK‐3β/β‐catenin pathway

Abstract: Glycogen synthase kinase-3beta (GSK-3beta) and adenomatous polyposis coli (APC) play an important role in the regulation of beta-catenin. Inhibition of or defects in their functions can lead to activation of beta-catenin. beta-catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF-kappaB). However, the regulatory roles of GSK-3beta/APC on the NF-kappaB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK-3beta/APC might regulate NF-kappaB activity through beta-catenin. We found that inhibition of GSK-3beta suppressed NF-kappaB activity, whereas reexpression of APC restored NF-kappaB activity in APC mutated cells. The regulatory effects were through beta-catenin because depletion of beta-catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF-kappaB target TRAF1 was inversely correlated with activated beta-catenin. Thus, APC/GSK-3beta, through beta-catenin, may crossregulate NF-kappaB signaling pathway.

Elevated expression of TGF-β1 in head and neck cancer-associated fibroblasts

Abstract: Head and neck cancers are characterized by a vigorous desmoplastic response, but the contribution of stromal-derived growth factors to the tumor microenvironment is poorly understood We evaluated the expression of stromal growth factor expression in head and neck squamous cell carcinoma (HNSCC) in normal and tumor-associated stromal cells Stromal tissue was isolated from epithelial cells with laser capture microdissection (LCMD) and analyzed by cDNA array for the expression of TGFalpha, TGF-beta1, HGF, PDGF-alpha, IGFII, bFGF, aFGF, VEGFC, and VEGF Primary fibroblasts were isolated in vitro from HNSCC tumors, adjacent histologically normal mucosa, and skin in vitro Fibroblast populations were assessed for TGF-beta1 expression by ELISA and luciferase reporter assay to assess protein expression We identified TGF-beta1 and IGFII overexpression in normal and tumor-associated stromal cells; however, only TGF-beta1 was significantly overexpressed (34-fold) in tumor-associated stroma Assessment of carcinoma-associated fibroblasts (CAFs), normal dermal fibroblasts (NDFs), and normal mucosal fibroblasts (NMFs) in propagated fibroblasts demonstrated persistently elevated levels of TGF-beta1 in CAFs compared to NMF and NDF populations Elevated levels of TGF-beta1 were identified in the stromal compartment of HNSCC tumors compared to normal mucosa by immunohistochemical analysis These results suggest that TGF-beta1 mRNA and protein is specifically upregulated in CAFs in vitro and in vivo

Relative amounts of antagonistic splicing factors, hnRNP A1 and ASF/SF2, change during neoplastic lung growth: implications for pre-mRNA processing.

Abstract: Pre-mRNA processing is an important mechanism for globally modifying cellular protein composition during tumorigenesis. To understand this process during lung cancer, expression of two key pre-mRNA alternative splicing factors was compared in a mouse model of early lung carcinogenesis and during regenerative growth following reversible lung injury. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and alternative splicing factor/splicing factor 2 (ASF/SF2) act antagonistically to modulate splice site selection. Both hnRNP A1 and ASF/SF2 contents rose in adenomas and during injury-induced hyperplasia compared to control lungs, as measured by immunoblotting. While both proteins increased similarly during compensatory hyperplasia, hnRNP A1 increased to a much greater extent than ASF/SF2 in tumors, resulting in a 6-fold increase of the hnRNP A1 to ASF/SF2 ratio. Immunohistochemical analysis showed that hnRNP A1 localized exclusively within tumor nuclei, while ASF/SF2 appeared in cytoplasm and/or nuclei, depending on the growth pattern of the tumor cells. We also demonstrated cancer-associated changes in the pre-mRNA alternative splicing of CD44, a membrane glycoprotein involved in cell-cell and cell-extracellular matrix interactions. hnRNP A1 and ASF/SF2 expression is thus differentially altered in neoplastic lung cells by mechanisms that do not strictly arise from increased cell division. These changes are influenced by tumor histology and may be associated with production of variant CD44 mRNA isoforms. © 2004 Wiley-Liss, Inc.

Epigenetic and gene expression changes related to transgenerational carcinogenesis

Abstract: Transgenerational carcinogenesis refers to transmission of cancer risk to the untreated progeny of parents exposed to carcinogens before mating. Accumulated evidence suggests that the mechanism of this process is epigenetic, and might involve hormonal and gene expression changes in offspring. To begin to test this hypothesis, we utilized a mouse model (NIH Swiss) in which exposure of fathers to Cr(III) chloride 2 wk before mating can alter incidence of neoplastic and nonneoplastic changes in offspring tissues. Utilizing a MS-RDA approach, we found that the sperm of these fathers had a significantly higher percentage of undermethylated copies of the 45S ribosomal RNA gene (rRNA); this finding was confirmed by bisulfite sequencing. Because gene methylation is a known mechanism of expression control in germ cells, and ribosomal RNA levels have been linked to cancer, these findings are consistent with the hypothesis. Secondly, we observed that offspring of Cr(III)-treated fathers were significantly heavier than controls, and had higher levels of serum T3. Possible effects of T3 levels on gene expression in the offspring were examined by microarray analysis of cDNAs from liver. A total of 58 genes, including 25 named genes, had expression ratios that correlated significantly with serum T3 ratios at P

Suppression of polyamine catabolism by activated Ki-ras in human colon cancer cells.

Abstract: An activated Ki-ras was expressed in the human colon adenocarcinoma cell line Caco-2 to study the effects of Ki-ras oncogene on polyamine metabolism during gastrointestinal tumorigenesis. Multiple clones selected for expression of the mutant Ki-ras transgene displayed a suppression of transcription of a key catabolic enzyme in polyamine catabolism spermidine/spermine N1-acetyltransferase (SSAT). Gene expression analysis, with cDNA microarrays, showed that Ki-ras transfected clones had decreased levels of expression, compared to mock transfected cells, of peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation. The activated Ki-ras suppressed SSAT expression by a mechanism involving the PPARgamma response element (PPRE) located at +48 bp relative to the transcription start site of the SSAT gene. Transient expression of the PPARgamma protein in Ki-ras expressing Caco-2 clones, or treatment with the PPARgamma ligand ciglitazone, led to an increase in the SSAT promoter activity. A MEK1/2 inhibitor PD98059 induced transcription of both PPARgamma and SSAT genes in the activated Ki-ras clones, suggesting that the mitogen-activated protein kinases (MAPKs) were involved in the regulation of SSAT expression by PPARgamma. We concluded that mutated Ki-ras suppressed SSAT via a transcriptional mechanism involving the PPARgamma signaling pathway.

Differential protein profiling in renal-cell carcinoma.

Abstract: Characterizing the alterations of protein expression in cancer cells can be very useful in providing insight into the changes in the functional pathways and thus the fundamental mechanisms of cancer development at the molecular level. In this study, we profiled protein expressions in eleven pairs of primary cell cultures derived from renal-cell carcinoma (RCC) tissues and patient-matched normal kidney tissues utilizing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Together with the immunoblot analysis of proteins from the RCC tissues, the study also demonstrated that the alterations of protein expression observed in RCC primary cell cultures reflected those observed in the original RCC tissues. We analyzed the expression profiles and identified proteins differentially expressed in RCC primary cell cultures by 2-D PAGE and mass spectrometry (MS). We found sixteen proteins were overexpressed and seven proteins underexpressed in RCC. The deregulated expressions of proteins include those involved in metabolism, cellular morphology, heat shock response, cell growth, etc. Overexpression of three proteins, αβ-crystallin, manganese superoxide dismutase (MnSOD), and annexin IV, most commonly observed in primary RCC cell cultures, were also observed by immunoblot analysis of proteins from the RCC tissues from which the primary cell cultures were derived. Semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis revealed the direct correlation between deregulated gene expression and the corresponding protein abundance in two of the three most commonly upregulated proteins we found in RCC. © 2004 Wiley-Liss, Inc.

Using DNA microarray analyses to elucidate the effects of genistein in androgen-responsive prostate cancer cells: identification of novel targets.

Abstract: Many studies have correlated the consumption of soy-rich diets with a decreased risk of developing hormone-dependent cancers, including prostate cancer. Genistein is a candidate prostate cancer preventive phytochemical found at high levels in soybean and soy foods. To better understand the molecular mechanisms underlying the beneficial effects of genistein on prostate cancer prevention, we used a DNA microarray approach to examine the effects of genistein at concentrations in the physiologic range on global gene expression patterns in androgen-responsive cancer cells. Microarray analyses were performed on androgen-responsive LNCaP human prostate cancer cells exposed to 0, 1, 5, or 25 μM genistein. We found a concentration-dependent modulation of multiple cellular pathways that are important in prostate carcinogenesis. Interestingly, the androgen receptor (AR)-mediated pathways, in particular, appeared to be modulated by genistein at the lowest concentrations. Based on these results, we propose that the regulation of AR-mediated pathways is potentially the most relevant chemopreventive mechanism for genistein administered at physiologic levels. Published 2004 Wiley-Liss, Inc.

Tcf binding sequence and position determines β-catenin and Lef-1 responsiveness of MMP-7 promoters

Abstract: The matrix metalloproteinase-7 (MMP-7) gene is a target of beta-catenin transactivation. Expression of the T-cell factor, Lef-1, enhances transcriptional activation of the human MMP-7 promoter by beta-catenin, but represses activation of the mouse MMP-7 promoter, both activities through consensus Tcf binding sites. The mouse promoter has a single Tcf binding element (mTBE) located downstream of the transcriptional start site, while the human promoter has two Tcf binding elements (hTBE1, hTBE2), both located upstream of the transcriptional start. hTBE1 and hTBE2 also differ in sequence from mTBE. Here we demonstrate that positioning of mTBE, upstream or downstream of the transcriptional start site dictated whether Lef-1 functioned as an activator or repressor, respectively. Sequence differences between mTBE and hTBE sites determined the potency of these activities, with hTBE sites being weaker. Mutational analysis of mTBE showed that increased Lef-1 activity mapped to G . C base pairings at 5' and 3' ends, and correlated with a threefold increase in Lef-1 binding affinity in vitro. Heterologous promoters with high affinity binding sites were 115-fold more responsive to beta-catenin than those with low affinity sites. Converting low affinity Tcf binding sites to high affinity sites increased beta-catenin responsiveness of the mouse and human promoters by 2-3 fold, and ectopic expression of Lef-1 increased beta-catenin responsiveness for promoters with low affinity binding sequences. We concluded that sequence and position of Tcf binding sites can determine the extent of beta-catenin-Lef-1 responsiveness for beta-catenin target genes.

Chronic expression of RET/PTC 3 enhances basal and insulin-stimulated PI3 kinase/AKT signaling and increases IRS-2 expression in FRTL-5 thyroid cells.

Abstract: The RET/PTC3 oncogene is a genetically rearranged and constitutively activated tyrosine kinase receptor that is common in papillary thyroid cancer. Because RET/PTC3 is chronically overexpressed in these thyroid cancer cells, and RET/PTC3-expressing tumors are associated with overactivity of tyrosine kinase signaling pathways and a more aggressive clinical course, we questioned whether chronic RET/PTC3 expression enhances cellular responses to thyroid mitogens in vitro. We stably transfected FRTL-5 cells with the RET/PTC3 gene; transfected and control cell lines were cultured without insulin, TSH, or serum. Thymidine incorporation into DNA was enhanced in the RET/PTC3 cells, but transformation was not observed. RET/PTC3 cells demonstrated higher basal and insulin-stimulated levels of activated Akt, both of which were reduced by LY294002, a PI3 kinase inhibitor, but not PD98059, a MEK inhibitor. By contrast, mitogen activated protein kinase (MAP kinase) was only minimally activated in RET/PTC3 cells before and after stimulation. Consistent with preferential activation of PI3 kinase, increased levels of total and phosphorylated IRS2 protein, relative activation of PDK-1, and enhanced IRS2-p85 interactions were identified in RET/PTC3-expressing cells. RET/PTC3 cells were also sensitized to insulin-induced thymidine incorporation; this effect was blocked by PI3 kinase (LY294002) rather than MEK 1/2 (PD98059) inhibitors. In summary, we have demonstrated that RET/PTC3 expression enhances basal and insulin-stimulated DNA synthesis through PI3 kinase, cooperatively activates Akt with insulin via PI3 kinase, and preferentially activates the Akt rather than MAP kinase pathway in FRTL-5 cells.

Microarray analysis of prostate cancer progression to reduced androgen dependence: Studies in unique models contrasts early and late molecular events

Abstract: Three unique variants of the CWR22 human prostate cancer xenograft model (CWR22LD1, LD2, and LD3) with a decrease in dependence on androgens were selected under noncastrate conditions, i.e., by outgrowth after transplantation into male NCR (AT) nu mice without testosterone supplementation. These variants were unable to grow in castrated male mice. For comparison, a second set of variants with even less dependence on androgens (castrate-resistant) were derived following outgrowth from CWR22 (CWR22Rv1 and RC) or CWRLD1 (CWR22RS) after transplantion in castrated male mice. The androgen receptor (AR) gene in the CWR22LD variants was transcriptionally active and was neither mutated nor significantly overexpressed compared to CWR22. Oligonucleotide microarray analysis showed distinctly different profiles of dysregulated gene expression among the CWR22LD variants. Groups of only 26-41 genes were dysregulated greater than threefold with a different proportion of up versus downregulated genes in each variant. Only one of the castrate-resistant variants (CWR22Rv1) had a highly overexpressed AR gene but AR in this variant and the two other castrate-resistant variants, CWR22 RS and RC, was not mutated beyond that seen in CWR22. In contrast to the CWR22LD variants, a total of 342, 295, and 222 genes were dysregulated at least threefold in CWR22Rv1, CWR22RS, and CWR22RC, respectively, differing as well in the proportion of up versus downregulated genes. Many of the genes dysregulated in CWR22LD1, LD2, and LD3 were further dysregulated in CWR22Rv1, RC, or RS. The most downregulated gene was microseminoprotein beta (MSPB). Along with cyclin D1, the most upregulated gene by an order of magnitude compared to other upregulated genes was hepatocyte growth factor (HGF) (scatter factor). These results suggest that the onset in the loss of androgen dependence in CWR22 proceeds through multiple pathways and does not require any direct change in the status of AR. However, upregulation of other survival pathways like that involving HGF in these studies could co-activate AR signaling. The endogenous overexpression of genes regulating sterol biosynthesis also observed in castrate-resistant CWR22 variants delineated a clinically relevant, compensatory mechanism for overcoming androgen deprivation reaffirming a central role for AR signaling in this process.

Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism.

Abstract: Ultraviolet (UV) light-induced activation of activator protein-1 (AP-1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP-1 activation. We have, therefore, examined the effects of two well-known inducers of phase II enzymes, sulforaphane (SF) and tert-butylhydroquinone (tBHQ), on UVB-induced AP-1 activation, with an AP-1-luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase-1 (QR-1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1-10 microM SF or 11-45 microM tBHQ for 24 h resulted in up to 1.4-fold and 1.7-fold increase of QR-1 activity, respectively, and up to 1.5-fold and 1.6-fold increases in cellular GSH levels, respectively. AP-1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m(2) of UVB. While the above SF treatment dose-dependently reduced the UVB-induced AP-1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB-induced AP-1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB-induced AP-1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP-1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB-induced AP-1 activation. The inhibitory effect of SF on UVB-induced AP-1 activation appears to be at least partly due to the direct inhibition of AP-1 DNA binding activity. This direct effect of SF on AP-1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP-1 activation.

The chemopreventive agent α-difluoromethylornithine blocks ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells

Abstract: Mutation of the Kirsten-ras (Ki-ras) proto-oncogene occurs frequently in colorectal cancers α-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki-ras transformation and colon tumorigenesis in carcinogen-treated animal models by mechanisms yet to be elucidated Caco-2 cells transfected with an activated Ki-ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice DFMO treatment (2% in drinking water) prevented tumor growth Gene expression profiling was performed to identify Ki-ras–and DFMO-dependent patterns of gene expression Microarray results were validated with real-time or semi-quantitative RT-PCR and/or Western blot analysis Genes upregulated in Caco-2 cells expressing an activated Ki-ras encoded cytoskeletal-, transport-, protease-, and gap junction–associated proteins These genes are important for normal development and maintenance of colonic epithelial tissue Caco-2 cells transfected with an activated Ki-ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin These parameters were unaffected by DFMO, but Ki-ras–dependent migration was inhibited by INGA1 antibodies Other Ki-ras–dependent, but DFMO-independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6) Ki-ras–transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1 DFMO reversed these increases The results indicated that the Ki-ras oncogene caused changes in experimental cell migration and cell-cell communication genes and that some of these changes could be reversed by DFMO © 2004 Wiley-Liss, Inc

Identification of novel differentially expressed genes by the effect of a high-fat n-6 diet in experimental breast cancer.

Abstract: In previous studies, we demonstrated that high corn oil diets promote the development of 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary tumors. In this study, we have investigated whether modulation of gene expression is one of the mechanisms by which this high-fat diet exerts such effects. Female Sprague-Dawley rats were induced with DMBA and fed normolipidic (3% corn oil) or high-fat (20% corn oil) diet. Screening of genes differentially expressed in adenocarcinomas from the high corn oil diet group compared to the control diet group was performed with cDNA microarrays. The resulting six upregulated and nine downregulated genes were validated by Northern blot and/or reverse transcription (RT)-polymerase chain reaction (PCR). Further investigation in a higher number of adenocarcinomas showed that in the high-fat n-6 diet group, where the tumor phenotype was verified to be more aggressive, the expression of submaxillary gland α-2u globulin, vitamin D3-upregulated protein 1 (VDUP1), H19, and the unknown function gene that codifies the expressed sequence tag (EST)-Rn.32385 was significantly decreased in comparison with the control group (C). These results, together with the fact that VDUP1, H19, and this globulin have been associated with cell proliferation and differentiation, open a new line of research about how the underexpression of these genes contributes to the stimulating effect of a high corn oil diet on experimental mammary carcinogenesis. © 2004 Wiley-Liss, Inc.

Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells.

Abstract: Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild-type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1-blocked ER-positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1-blocked ER-positive BG-1 ovarian cancer cells. For all three drugs, BRCA1-blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 microM in the control cells and 2.68 microM for the BRCA1-blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild-type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1-mutated ovarian cancer patients.

CHFR-associated early G2/M checkpoint defects in breast cancer cells.

Abstract: Cell division is a highly regulated process. Checkpoints can halt cell-cycle progression due to adverse conditions such as misalignment of chromosomes to prevent missegregation. The search for new regulators of the cell cycle revealed the mitotic checkpoint gene CHFR (checkpoint with forkhead-associated and ring finger). CHFR coordinates an early mitotic phase by delaying chromosome condensation in response to a mitotic stress. Because aneuploidy and chromosome instability are common in malignant breast tumors, we screened 24 breast cancer cell lines for CHFR expression and demonstrated that 50% (12 of 24) of breast cancer cell lines had low CHFR levels. Expression of CHFR was reactivated with the demethylating agent 5-aza-2 0 -deoxycytidine (5-aza-dC) in two low-CHFR–expressing cell lines. Eleven of these 12 (92%) low-CHFR–expressing cell lines had an unusually high number of condensed chromosomes and high mitotic indices in response to nocodazole treatment. Transfection of CHFR in one of these cancer cell lines lowered the mitotic index after nocodazole treatment. In conclusion, our data suggested that low CHFR expression associated with high mitotic indices in response to nocodazole treatment were common in the breast cancer cell lines studied. Additional flow cytometry studies and analysis of a protein that interacts with CHFR in vitro, polo-like kinase 1 (PLK1), suggests that this CHFR-associated early G2/M checkpoint is complex, involving additional, as yet unidentified, proteins. Further analysis of CHFR in breast cancer cells will be important for understanding the complex mechanisms leading to aneuploidy and chromosomal instability observed in breast cancer. 2003 Wiley-Liss, Inc.

Coregulators and chromatin remodeling in transcriptional control

Abstract: Despite many years of investigation by numerous investigators, transcriptional regulatory control remains an intensely investigated and continuously evolving field of research. Transcriptional regulation is dependent not only on transcription factor activation and chromatin remodeling, but also on a host of transcription factor coregulators-coactivators and corepressors. In addition to transcription factor activation and chromatin changes, there is an expanding array of additional modifications that titrate transcriptional regulation for the specific conditions of a particular cell type, organ system, and developmental stage, and such events are likely to be greatly influenced by upstream signaling cascades. Here, we will briefly review the highlights and perspectives of chromatin remodeling and transcription controls as affected by cofactor availability, cellular energy state, relative ratios of reducing equivalents, and upstream signaling. We also present the C-terminal binding protein (CtBP) as a novel nuclear receptor (NR) coregulator, which exemplifies the integration of a number of transcriptional regulatory controls.

Negative growth control of renal cell carcinoma cell by connexin 32: possible involvement of Her-2.

Abstract: Connexin (Cx) genes have negative growth effects on tumor cells with certain cell specificity. We have previously reported that Cx32 is specifically downregulated in human renal cell carcinoma cell (RCC) lines as well as cancerous regions of kidneys and that the Cx is expressed in the progenitor cells of the carcinoma. However, the precise role of Cx32 in growth control of RCC cells remains unknown. In this study, we examined whether Cx32 could act in growth control against a human RCC cell, Caki-2 cell. In order to estimate the cell growth control, we established Caki-2 cells that have stable expression of Cx32 genes. Cx32 expression in Caki-2 cells induced contact inhibition of growth and reduced anchorage-independent growth ability, but did not significantly affect lag phase growth rates. This growth control by Cx32 was dependent on the inhibition of the cell-cycle transition from G1 to S phase at high cell density, and the inhibition of the cell-cycle transition related to the suppression of Her-2 activation. Furthermore, the suppression of Cx32 expression in Caki-2 cells by short interfering RNA induced the activation of Her-2. These data suggest that Cx32 has negative growth control of Caki-2 cells, partly due to the inhibition of the Her-2 activation.

Expression profile of eukaryotic translation factors in human cancer tissues and cell lines

Abstract: Several studies have demonstrated the overexpression of certain eukaryotic translation factors in human cancer cell lines and in malignant tissues. In this study, with human cancer cell lines derived from lungs, breast, prostate, and skin, we have examined the expression profile of 36 translation factors consisting of 27 initiation factors, 8 elongation factors, and 1 termination factor. Translation initiation factors 2C2 and 4E1 and translation elongation factors 1A2 and 1delta were found overexpressed (2- to 2000-fold) in many of the cancer cell lines compared to their corresponding normal cell lines. Among the translation factors analyzed, translation elongation factor 1A2 exhibited the most significant alteration in expression: 10- to 2000-fold overexpression was noticed in nine out of ten cancer cell lines analyzed. Whether the overexpression of translation elongation factor 1A2 can be used as a potential tumor marker was tested with the cancer profiling array (BD Biosciences, Palo Alto, CA) consisting of 241 paired cDNA samples generated from 13 different cancer/noncancer tissue types. Overexpression of translation elongation factor 1A2 was noticed in several tumor tissue samples, most notably in the human colon cancer samples which exhibited at least a twofold overexpression among 35% of the samples analyzed. Besides colon, tumor samples derived from lungs, kidney, rectum, and ovary also exhibited more than a twofold overexpression of translation elongation factor 1A2 in at least 20% of the samples analyzed. These results indicate that human carcinogenesis is often associated with alterations in the expression of various translation factors especially the overexpression of eukaryotic translation elongation factor 1A2.

Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-κB in mouse epidermal cl41 cells†

Abstract: Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs.

The molecular mechanism of sensitization to Fas‐mediated apoptosis by 2‐methoxyestradiol in PC3 prostate cancer cells

Abstract: It is widely known that death receptor Fas-dependent apoptotic signals are associated with development of prostate cancer, but the key pathways involved in sensitivity to the apoptosis remain unclear. Here we investigated the molecular mechanism by which 2-methoxyestradiol (2-ME) effectively sensitizes a human prostate cancer cell line, PC3, to Fas-mediated apoptosis. 2-ME significantly inhibited nuclear factor-kappaB (NF-kappaB) activation and downregulated Fas-associated death domain (FADD) protein interluekin-1beta-converting enzyme inhibitory protein (FLIP). Overexpression of the dominant negative mutant form of IkappaBalpha (d/n IkappaBalpha) or treatment with Ikappa kinase-specific inhibitor Bay117082 gave the same results, although the sensitizing effect was not as pronounced. A selective inhibitor of Akt phosphorylation, LY294002, accelerated formation of the death-inducing signaling complex (DISC) not only by FLIP reduction but also by enhancement of recruitment of the FADD to Fas, thereby sensitizing PC3 cells to apoptosis similar to the case with 2-ME stimulation. Moreover, we found that inhibition of 2-ME-induced extracellular signal-regulated kinase (ERK) activation by the upstream kinase inhibitor PD98059 significantly enhanced 2-ME-mediated suppression of Akt activation, resulting in much greater sensitization to apoptosis. Taken together, the present findings indicate that 2-ME suppresses NF-kappaB/FLIP signaling and enhances DISC formation through inhibition of Akt, and that PC3 cells thereby are being sensitized to Fas-mediated apoptosis and by a process closely associated with ERK.

Estrogen-dependent regulation of ornithine decarboxylase in breast cancer cells through activation of nongenomic cAMP-dependent pathways.

Abstract: 17β-Estradiol (E2) induces ornithine decarboxylase (ODC) activity in several E2-responsive tissues/cells, and this study investigated the mechanism of hormone-induced transactivation in MCF-7 human breast cancer cells. E2-induced reporter gene (luciferase) activity in MCF-7 cells transfected with a construct (pODC1) containing the −164 to +29 region of the human ODC gene promoter linked to bacterial luciferase. This promoter sequence contains GC-rich Sp1 binding sites, CAAT, LSF, cAMP response element (CRE), and TATA motifs. Deletion and mutational analysis of the ODC promoter showed that both CAAT and LSF sites were required for hormone-induced transactivation. Gel mobility shift and DNA footprinting assays indicated that NFYA and LSF bound the CAAT and LSF motifs, respectively, and GAL4-NFYA/GAL4-LSF chimeras were also activated by E2, 8-bromo-cAMP, and protein kinase A (PKA) expression plasmid. However, E2-induced transactivation of GAL4-NFYA and GAL4-LSF was blocked by the PKA inhibitor SQ22356 indicating that the mechanism of ODC induction by E2 involves upregulation of cAMP/PKA through nongenomic pathways of estrogen action. © 2004 Wiley-Liss, Inc.

LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane.

Abstract: Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras-transformed 1-1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C-terminal truncated form of LyGDI (Delta C-LyGDI, 166-201 deletion), a member of the RhoGDIs. The stable expression of Delta C-LyGDI induced pulmonary metastasis in 1-1ras1000 cells, whereas expression of full-length LyGDI did not induce metastasis. Delta C-LyGDI was preferentially localized in the membrane, detected in a NP-40-insoluble fraction, and co-purified with radixin, moesin, Rac1, Cdc42, and RhoA. In Delta C-LyGDI transfectant, an activation state of Rac1 was elevated and Delta C-LyGDI was associated with Rac1-GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1-GTP, Delta C-LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis.

Human S15a expression is upregulated by hepatitis B virus X protein.

Abstract: The hepatitis B virus (HBV)-encoded X antigen (HBxAg) may contribute to the development of hepatocellular carcinoma (HCC) through the upregulated expression of selected cellular genes. To identify these genes, RNAs isolated from HBxAg-positive and -negative HepG2 cells were compared by PCR select cDNA subtraction. One gene overexpressed in HBxAg-positive cells by Northern and Western blotting is the ribosomal protein S15a. The S15a mRNA is 535 base pairs, encoding a protein 130 amino acids long with a molecular weight of 14.3 kDa. S15a expression was upregulated in HBV-infected livers, where it costained with HBxAg. Overexpression of S15a stimulated cell growth, colony formation in soft agar, and tumor formation in SCID mice. Hence, HBxAg upregulated the expression of S15a, the latter of which participates in the development of HCC, perhaps by altering the integrity of translation.