YY1 negatively regulates the XAF1 gene expression in prostate cancer.

XAF1 is a tumor suppressor gene with low or absent expression in cancer. Since transcriptional reactivation or ectopic-mediated expression of XAF1 inhibits tumor growth, it is of great interest to elucidate the molecular mechanisms leading to XAF1 silencing. YY1 is a transcription factor that acts as a repressor or an activator to modulate several cancer-associated cellular processes. Both YY1 and XAF1 have key roles in prostate cancer (PCa) progression and are associated with worse clinical outcomes. To assess whether YY1 regulates the transcriptional activation of the XAF1 gene, we performed gene-reporter assays coupled with site-directed mutagenesis, which showed that YY1 is able to mediate XAF1 silencing. Concordantly, ChIP-qPCR assays showed that YY1 interacts with the XAF1 promoter in PC3 cells that lacks XAF1 expression. This association was lost after exposure to epigenetic modulators that induce XAF1 expression. Further supporting the YY1's repressive role, we found transcriptional reactivation of the XAF1 gene by YY1 downregulation. As expected by previous reports showing that HDAC1 is needed for YY1-mediated repressive actions, we observed XAF1 re-expression after either inhibition or downregulation of the HDAC1 gene. Finally, expression data retrieved from the TCGA consortium showed that PCa samples presented lower XAF1 and higher HDAC expression levels than normal tissues. Thus, our results support a model in which YY1 is able to silence tumor suppressor genes such as XAF1 through HDAC1 in PCa.

TIMP4 Modulates ER-α Signalling in MCF7 Breast Cancer Cells.

Tissue inhibitor of metalloprotease 4 (TIMP4) contributes to poor prognosis in breast and other tumours. However, the mechanisms of how TIMP4 influences breast cancer cell behaviour are unknown. Our aim was to explore the signalling pathways modulated by TIMP4 in breast cancer cells. Human recombinant TIMP4 was added to MCF7 breast cancer cells and RNASeq was performed. TIMP4 RNASeq results were validated by RT-PCR. Network analyses of TIMP4-exposed cells showed that ER-α, HIF1A and TGF-ß signalling were activated, whereas FOXO3 signalling was downregulated. ER-α protein levels were increased and concordantly, promoters of TIMP4-upregulated genes were significantly enriched in oestrogen-binding sites. We concluded that TIMP4 modulates multiple signalling pathways relevant in cancer in MCF7 cells, including the ER-α cascade.

Renin is an angiotensin-independent profibrotic mediator: role in pulmonary fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is probably the result of interplay between cytokines/chemokines and growth factors. The renin-angiotensin (Ang) system is involved, although its profibrotic effect is attributed to Ang II. However, recent studies suggest that renin, through a specific receptor, is implicated in fibrogenesis. In this study, the expression of renin and renin receptor was examined in normal and IPF lungs and fibroblasts. Normal human lung fibroblasts were stimulated with renin or transfected with renin small interfering RNA (siRNA), and the expression of transforming growth factor (TGF)-ß1 and α-1-type I collagen was analysed. Normal lungs and lung fibroblasts expressed renin, which was strongly upregulated in IPF lungs and fibroblasts (∼10-fold increase; p<0.05). Immunocytochemistry showed intense renin staining in IPF fibroblasts. Renin-stimulated lung fibroblasts displayed an increase in the expression of TGF-ß1 (mean ± sd 1.8 × 10(3) ± 0.2 × 10(3) versus 1.2 × 10(3)± 0.3 × 10(3) mRNA copies per 18S ribosomal RNA; p<0.01) and collagen (5.93 × 10(2)± 0.66 × 10(2) versus 3.28 × 10(2) ± 0.5 × 10(2); p<0.01), while knocking down renin expression using siRNA provoked a strong decrease of both molecules. These effects were independent of Ang II, since neither losartan nor captopril decreased these effects. Renin also decreased matrix metalloprotease-1 expression and induced TGF-ß1 activation (163 ± 34 versus 110 ± 15 pg active TGF-ß1 per mg total protein). These findings highlight the possible role of renin as an Ang II-independent profibrotic factor in lung fibrosis.

Gene regulation by BCL3 in a cervical cancer cell line.

BCL3 is a putative proto-oncogene deregulated in haematopoieitic and solid tumours. It has been suggested that its oncogenic effects could be mediated, at least in part, by inducing proliferation and inhibiting cell death. To provide more insight into the mediators of these effects, we used an unbiased approach to analyse the mRNA expression changes after knocking-down BCL3 using specific shRNAs. One hundred eighty genes were up-regulated and sixtynine genes were down-regulated after knocking down BCL3. Function analyses showed enrichment in genes associated with cellular growth and proliferation, cell death and gene expression. We found that STAT3, an important oncogene in human cancer, was the central node of one of the most significant networks. We validated STAT3 as a bona fide target of BCL3 by additional interference RNA and in silico analyses of previously reported lymphoma patients.

NF-kappa B is required for the development of tumor spheroids.

Tumor cells cultured in three-dimensional models provide a more realistic and biologically meaningful analysis of the initial phases of cancer development and drug resistance. Several studies have demonstrated that culture of cancer cells in three dimensions induces cellular resistance to a variety of anti-neoplastic drugs by poorly understood mechanisms. The role of the transcription factor NF-kappaB and inhibitors of apoptosis proteins (IAPs) in the onset and development of drug resistance during tumor spheroid growth has not been established. In this work, we found a significant increase in the activity and expression of NF-kappaB and its downstream target XIAP (X-linked IAP) in cancer cells grown as multi-cellular tumor spheroids. Blocking XIAP expression with RNA interference markedly increased the sensitivity of cancer tumor spheroid cells toward anti-neoplastic drugs, indicating a role for IAPs in establishing drug resistance. In turn, inhibition of NF-kappaB by negative dominants suppressed spheroid formation, whereas overexpression of the upstream kinase IkappaBKbeta increased their growth and resistance. The present data suggested that NF-kappaB and its downstream target XIAP were essential for the growth and drug resistance of small avascular tumor.

Tumor HGF lacks prognostic significance in Mexican breast cancer patients.

Hepatocyte growth factor-scatter factor (HGF-SF) is a pleiotropic protein implicated in tumor formation and metastasis. Since increased levels of HGF-SF were first identified in breast cancer tissue or patient serum, some evidence has suggested that tumor or serum HGF-SF concentration could be one of the most accurate prognostic factors for this disease. However, other recent investigations have not been able to corroborate this finding. The study aims to establish the prognostic significance of HGF-SF in Mexican breast cancer women. Surgical specimens were obtained from 67 incident breast cancer patients at the Mexican National Cancer Institute between 1994 and 1995. Primary breast cancer tissue HGF-SF was measured by quantitative sandwich enzyme immunoassay. Relapse-free and overall survival curves were generated using Kaplan-Meier method. Significance of survival differences was calculated by log-rank test. chi2 was used for the association analysis between prognostic variables. Disease-free survival and overall survival were similar between the high tumor HGF group of patients and the low HGF patients (p = 0.7 and p = 0.36 respectively). No association was found between HGF and other clinicopathological variables (age, menopause status, clinical tumor size, clinical node involvement, metastasis, tumor grade, Estrogen Receptor and Progesterone Receptor). We found no prognostic significance for HGF, nor did we find a clear association between HGF and other known prognostic factors. A firm conclusion cannot be established regarding the role of HGF as a prognostic tool in breast cancer patients.

Benzene metabolites induce apoptosis in lymphocytes.

Benzene is an important environmental pollutant with important health implications. Exposure to this aromatic hydrocarbon is associated with hematotoxicity, and bone marrow carcinogenic effects. It has been shown that benzene induces oxidative stress, cell cycle alterations, and programmed cell death in cultured cells. Hepatic metabolism of benzene is thought to be a prerequisite for its bone marrow toxicity. Nevertheless, there are no reports on the cellular effects of reactive intermediates derived from hepatic metabolism of benzene. Thus, the goal of this project was to determine the cellular alterations of benzene metabolites produced by the cultured hepatic cell line HepG2. Supernatants collected from these cells were applied to a culture of freshly isolated lymphocytes. A higher decrease in cell viability was found in cells exposed to these supernatants than to unmetabolized benzene. This viability decrease was due to apoptosis, as determined by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and internucleosomal fragmentation of DNA. When supernatants were analyzed by HPLC, we found that not all the hydrocarbon was biotransformed, since a 28 microM concentration (37%) remained. The only metabolite found in the culture medium was muconic acid. The present results show that muconic acid derived from benzene metabolism is able to cooperate with the pollutant for the induction of apoptosis in rat lymphocytes.

NF-κB signaling in cancer stem cells: a promising therapeutic target?

BACKGROUND: Cancer stem cells (CSCs) are regulated by several signaling pathways that ultimately control their maintenance and expansion. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) forms a protein complex that controls DNA transcription and, as such, plays an important role in proliferation, inflammation, angiogenesis, invasion and metastasis. The NF-κB signaling pathway, which has been found to be constitutively activated in CSCs from a variety of cancers, participates in the maintenance, expansion, proliferation and survival of CSCs. Targeted disruption of this pathway may profoundly impair the adverse phenotype of CSCs and may provide a therapeutic opportunity to remove the CSC fraction. In particular, it may be attractive to use specific NF-κB inhibitors in chronic therapeutic schemes to reduce disease progression. Exceptional low toxicity profiles of these inhibitors are a prerequisite for use in combined treatment regimens and to avoid resistance. CONCLUSION: Although still preliminary, recent evidence shows that such targeted strategies may be useful in adjuvant chemo-preventive settings.

Inhibitor of apoptosis-1 (IAP-1) expression and apoptosis in non-small-cell lung cancer cells exposed to gemcitabine.

Exposure of lung cancer cells to gemcitabine (2',2'-difluorodeoxycytidine) arrests cells in S phase and induces secondary apoptotic cell death. Gemcitabine treatment decreased the expression of IkappaB-alpha protein and, concomitantly, increased the activity of nuclear factor-kappaB (NF-kappaB) transcription factor, a known inhibitor of the apoptotic response. This increase was accompanied by a similar increment in the expression of inhibitor of apoptosis-1 (IAP-1) protein and mRNA, a caspase inhibitor responsive to NF-kappaB. These changes were important to the final destiny of the cells, since overexpression of a dominant negative version of IkappaB-alpha, which suppresses NF-kappaB activation, blocks the increase of IAP-1 protein and potentiates the action of gemcitabine. Additionally, overexpression of IAP-1 protein in A549 cells expressing the IkappaB-alpha mutant restored the initial sensitivity to gemcitabine and demonstrated that this protein was responsible for the inhibitory effect of NF-kappaB. These results support the notion of IAP-1 as an important antiapoptotic protein mediating sensitivity to deoxynucleotides analogs in non-small-cell lung cancer cells.

Gemcitabine activity in cervical cancer cell lines.

PURPOSE: Gemcitabine (2',2'-difluorodeoxycytidine) is an antineoplastic agent with activity against a variety of solid tumors. To investigate its in vitro activity toward cervical cancer, we exposed six cervical cancer cell lines to gemcitabine. METHODS: Combinational cytotoxic studies using viability tests and clonogenicity assays. RESULTS: Gemcitabine was cytostatic and cytotoxic in some of the lines at peak plasma concentrations similar to those achieved in clinical trials. Gemcitabine was also found to effectively synergize with cisplatin and showed a radiosensitizing effect in these cells. The cytotoxicity observed in sensitive cell lines was due to apoptosis, as demonstrated by DNA fragmentation assays. CONCLUSIONS: We recommend performing additional in vitro experimentation so that these results can be confirmed to support clinical trials of gemcitabine in cervical cancer patients both as first-line therapy and with concomitant radiation.

Modulation of NF-kappa B, and Bcl-2 in apoptosis induced by cisplatin in HeLa cells.

Cisplatin exposure induces apoptosis in HeLa cells. Since the interaction of this drug with DNA produces reactive oxygen species, we performed an analysis of the oxidative stress-responsive factors AP-1 and NF-kappa B. Although AP-1 levels were not modified during cisplatin exposure, electrophoretic mobility shift assays demonstrated an increase in NF-kappa B DNA binding activity that correlated with a decrease of the inhibitory protein I kappa B alpha and a specific relocalization of c-Rel, as assessed by immunoblotting and immunofluorescence. No changes in the levels or localization of p65 were found. Interestingly, I kappa B alpha relocalized to the nucleus, probably in order to regulate the binding of specific complexes. This process was accompanied by a decrease of the antiapoptotic protein Bcl-2, and a relocalization of p53 protein to the nucleus. Since HeLa cells lost most of their p53 protein due to a specific E6-dependent degradation, cisplatin could be inhibiting this degradation, since the p53 total levels were not increased during the exposure to the drug.

Cisplatin-induced apoptosis of HeLa cells. Effect of RNA and protein synthesis inhibitors, Ca2+ chelators and zinc.

HeLa cells exposed to cisplatin undergo cell death, presenting morphological and biochemical characteristics typical of apoptosis. In this study we demonstrate that this process is independent of RNA and protein synthesis, since it was not inhibited by actinomycin D or cycloheximide. These substances induced apoptosis by themselves, suggesting an unidentified short-lived inhibitor. The presence of Ca2+ chelators (EDTA and EGTA) did not have effect in this process, excluding the participation of extracellular Ca2+ access. Finally, zinc ions inhibited the low molecular weight DNA degradation and the apoptotic bodies production, but not cell death. These results provide an insight into the mechanism of action of one of the most used antineoplastic drug.

Cupressus lusitanica (Cupressaceae) leaf extract induces apoptosis in cancer cells.

A crude ethanolic extract of Cupressus lusitanica Mill. leaves demonstrate cytotoxicity in a panel of cancer cell lines. Cell death was due to apoptosis, as assessed by morphologic features (chromatin condensation and apoptotic bodies formation) and specific DNA fragmentation detected by in situ end-labeling of DNA breaks (TUNEL). The apoptotic cell death was induced timely in a dose-dependent manner. Despite the absence of changes in the expression levels of antiapoptotic protein Bcl-2, proapoptotic Bax protein variants omega and delta were increased. These results warrant further research of possible antitumor compounds in this plant.

Participation of Omi Htra2 serine-protease activity in the apoptosis induced by cisplatin on SW480 colon cancer cells.

Apoptosis is triggered by two interconnected pathways, extrinsic and intrinsic. The intrinsic pathway is activated by genomic stress promoting mitochondrial release of apoptotic proteins. One of these proteins is Omi/Htra2, a serine protease which inactivates Inhibitor of Apoptosis Proteins (IAPs). In the present work, we assessed the participation of Omi/Htra2 in the cell death induced by the chemotherapeutic drugs 5-fluorouracil (5-FU) and cisplatin (CDDP) in SW480 colon cancer cells. CDDP and 5-FU induced apoptosis mediated by the intrinsic pathway in colon cancer cells, as demonstrated by morphological analyses, mitochondrial cytochrome c release and cleavage of caspase 3. Omi/Htra2 was also released from mitochondria of cells exposed to these drugs, as demonstrated by immunofluorescence and western blot assays of subcellular fractions. Exposure of cells to the Omi/Htra2 serine protease inhibitor UCF-101 prevented death p<0.0001 and partially suppressed reproductive cell death of cells exposed to cisplatin p<0.05, but not to 5-FU p=0.49. From these experiments we show that Omi/Htra2 serine protease activity participates in the cell death induced by CDDP but not of 5-FU in colon cancer cells.

Regulation of mitochondrial Smac/DIABLO-selective release by survivin.

The intrinsic apoptotic pathway is characterized by the release of several mitochondrial intermembrane proteins into the cytosol of dying cells. It is unclear whether the release of these proteins follows a common or specific pathway. In the present report we show that survivin and, to a lesser extent, the survivin splice variant survivin DeltaEx3 regulate the specific liberation of second mitochondria-derived activator of caspase/direct IAP binding protein with low pI (Smac/DIABLO), an inhibitor of apoptosis proteins binding protein, during apoptosis induced by etoposide, a DNA damaging agent. This antineoplastic drug induced posttranscriptional upregulation of survivin and survivin DeltaEx3. In turn, mitochondrial survivin associated with Smac/DIABLO, delaying its release. In addition, cytosolic survivin also stabilized the cytosolic levels of released Smac/DIABLO. These results provide an explanation for the observed differences in the release of mitochondrial intermembrane proteins in various apoptotic models and present a new mechanism for the anti-apoptotic effects of survivin in cancer cells.

Subcellular redistribution of HSP72 protein during cisplatin-induced apoptosis in HeLa cells.

Exposure of HeLa cells to cisplatin results in the activation of apoptotic cell death. This drug induces DNA damage and generates reactive oxygen intermediates. Since cisplatin is highly reactive and binds to diverse proteins, it could create abnormal protein structures or nonspecific aggregates. For these reasons, we analyzed the expression and subcelullar distribution of hsp72, a heat-shock protein that enables cells under stress to cope with damaged proteins. We did not observe any changes in the expression of hsp72 protein, although, by immunofluorescence studies, we detected a dramatic redistribution of the protein. These results and its probable relevance in the drug-induced apoptotic phenomenon are discussed.

Paclitaxel-induced apoptosis in HeLa cells is serum dependent.

Exposure of HeLa cells to different concentrations of the antineoplastic drug paclitaxel resulted in a loss of cell viability that was dependent on the concentration and time of exposure to the drug. This phenomenon was associated with the appearance of nuclear morphology typical of apoptosis and DNA breakage into a "ladder" pattern of discrete fragments of nucleosomal size. The induction of cell death was dependent on the serum concentration of the culture media, repressed by pretreatment with a cAMP-dependent protein kinase (PKA) inhibitor, and enhanced by increasing the cell proliferation with previous exposure to a cAMP-analog and a protein kinase-C (PKC) inducer. The proliferative index modifies the effect of taxol on HeLa cells, probably by means of a more rapid accumulation of cells in the G2/M cycle blockage point, although a direct participation of PKA and PKC should not be excluded.

Mitochondrial changes during the apoptotic process of HeLa cells exposed to cisplatin.

HeLa cells undergo apoptosis after exposure to cisplatin. Since mitochondria have recently been proposed as a probable effector of this type of cell death, we performed an analysis using the fluorescent cation rhodamine 123, which is transported actively by this organelle. Cisplatin induces a decrease in the mitochondrial staining, as assessed by cytofluorometric analysis. Microscopic analysis demonstrated that this effect was accompanied by damage of the mitochondria. These features were not exclusive of cisplatin, as other antineoplasic agents (taxol, etoposide) elicited similar effects. These results point toward the notion of a general effect of antineoplasic drugs over the mitochondria during induction of apoptotic cell death.