Sara Huerta-Yepez

Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-kappa B and inhibition of Bcl-xl expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues and this prompted its potential therapeutic application in cancer. However, not all cancers are sensitive to TRAIL-mediated apoptosis and, therefore, TRAIL-resistant cancer cells must be sensitized first to become sensitive to TRAIL. Treatment of prostate cancer (CaP) cell lines (DU145, PC-3, CL-1, and LNCaP) with nitric oxide donors (e.g. (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate)) sensitized CaP cells to TRAIL-induced apoptosis and synergy was achieved. The mechanism by which DETANONOate mediated the sensitization was examined. DETANONOate inhibited the constitutive NF-κB activity as assessed by EMSA. Also, p50 was S-nitrosylated by DETANONOate resulting in inhibition of NF-κB. Inhibition of NF-κB activity by the chemical inhibitor Bay 11-7085, like DETANONOate, sensitized CaP to TRAIL apoptosis. In addition, DETANONOate downregulated the expression of Bcl-2 related gene (Bcl-xL) which is under the transcriptional regulation of NF-κB. The regulation of NF-κB and Bcl-xL by DETANONOate was corroborated by the use of Bcl-xL and Bcl-x κB reporter systems. DETANONOate inhibited luciferase activity in the wild type and had no effect on the mutant cells. Inhibition of NF-κB resulted in downregulation of Bcl-xL expression and sensitized CaP to TRAIL-induced apoptosis. The role of Bcl-xL in the regulation of TRAIL apoptosis was corroborated by inhibiting Bcl-xL function by the chemical inhibitor 2-methoxyantimycin A3 and this resulted in sensitization of the cells to TRAIL apoptosis. Signaling by DETANONOate and TRAIL for apoptosis was examined. DETANONOate altered the mitochondria by inducing membrane depolarization and releasing modest amounts of cytochrome c and Smac/DIABLO in the absence of downstream activation of caspases 9 and 3. However, the combination of DETANONOate and TRAIL resulted in activation of the mitochondrial pathway and activation of caspases 9 and 3, and induction of apoptosis. These findings demonstrate that DETANONOate-mediated sensitization of CaP to TRAIL-induced apoptosis is via inhibition of constitutive NF-κB activity and Bcl-xL expression.

Rituximab-Induced Inhibition of YY1 and Bcl-xL Expression in Ramos Non-Hodgkin’s Lymphoma Cell Line via Inhibition of NF-κB Activity: Role of YY1 and Bcl-xL in Fas Resistance and Chemoresistance, Respectively

Rituximab treatment of B non-Hodgkin’s lymphoma (NHL) cell lines inhibits the constitutive NF-κB activity and results in the sensitization of tumor cells to both chemotherapy and Fas-induced apoptosis. Cells expressing dominant active IκB or treated with NF-κB-specific inhibitors were sensitive to both drugs and Fas agonist mAb (CH-11)-induced apoptosis. Down-regulation of Bcl-xL expression via inhibition of NF-κB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL-overexpressing Ramos cells, Ramos hemagglutinin (HA)-Bcl-x, which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent; Ramos HA-Bcl-x cells were as sensitive as the wild type to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor yin-yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab or the NO donor DETANONOate or after transfection with YY1 small interfering RNA resulted in up-regulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two mechanisms underlying the chemosensitization and immunosensitization of B-NHL cells by rituximab via inhibition of NF-κB. The regulation of chemoresistance by NF-κB is mediated via Bcl-xL expression, whereas the regulation of Fas resistance by NF-κB is mediated via YY1 expression and activity. The potential clinical significance of these findings is discussed.

Chemotherapeutic drugs sensitize cancer cells to TRAIL-mediated apoptosis: up-regulation of DR5 and inhibition of Yin Yang 1

Several chemotherapeutic drugs in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) result in reversal of resistance to TRAIL-mediated apoptosis through up-regulation of DR5 expression. The promoter of DR5 has one putative binding site for the transcription repressor Yin Yang 1 (YY1), and thus, we hypothesized that the sensitizing drugs may inhibit YY1. We have found that treatment of tumor cells with various chemotherapeutic drugs inhibited nuclear factor-κB. We examined whether drugs also inhibit YY1 activity and whether YY1 inhibition correlates with up-regulation of DR5 expression and sensitization of cells to TRAIL-induced apoptosis. The TRAIL- and drug-resistant prostate carcinoma PC-3 cell line was treated with CDDP, VP-16, ADR, and vincristine. DR5 luciferase reporter constructs and small interfering RNA against YY1 were used to determine the role of YY1 in DR5 transcription. Pretreatment of PC-3 cells and other tumor cell lines with various chemotherapeutic drugs sensitized the cells to TRAIL-induced apoptosis concurrently with up-regulation of DR5 expression and inhibition of YY1 expression and its DNA-binding activity. The baseline luciferase activity in PC-3 cells transfected with the wild-type DR5 reporter was significantly augmented in cells transfected with DR5 constructs carrying deletions or mutation in the YY1-binding site. Treatment with drug enhanced DR5 wild-type luciferase activity, with no increase in cells transfected with the YY1-deleted or YY1-mutated constructs. Cells transfected with YY1 small interfering RNA showed up-regulation of DR5 expression and sensitization to TRAIL-mediated apoptosis. The findings provide evidence that drug-induced sensitization of tumor cells to TRAIL is mediated, in part, by inhibition of the transcription repressor YY1 and up-regulation of DR5 expression. Hence, YY1 may be a potential therapeutic target to reverse resistance to TRAIL-induced apoptosis. [Mol Cancer Ther 2007;6(4):1387–99]

Rituximab (chimeric anti-CD20) sensitizes B-NHL cell lines to Fas-induced apoptosis.

Rituximab (chimeric anti-CD20 monoclonal antibodies) is currently being used in the treatment of B non-Hodgkins lymphoma (NHL). We have recently reported that rituximab triggers and modifies various intracellular signaling pathways in NHL B-cell lines, resulting in reverting the chemoresistant phenotype to a sensitive phenotype. This study investigated whether rituximab also modifies intracellular signaling pathways resulting in the sensitization of NHL cells to Fas-induced apoptosis. Treatment of the Fas-resistant NHL cell lines (2F7, Ramos and Raji) with rituximab sensitized the cells to CH-11 (FasL agonist mAb)-induced apoptosis and synergy was achieved. Fas expression was upregulated by rituximab as early as 6 h post-treatment as determined by flow cytometry, reverse transcriptase–polymerase chain reaction and Western blot. Rituximab inhibited both the expression and activity of the transcription repressor Yin-Yang 1 (YY1) that negatively regulates Fas transcription. Inhibition of YY1 resulted in the upregulation of Fas expression and sensitization of the tumor cells to CH-11-induced apoptosis. The downregulation of YY1 expression was the result of rituximab-induced inhibition of both the p38 mitogen-activated protein kinase (MAPK) signaling pathway and constitutive nuclear factor κ of B cells (NF-κB) activity. The involvement of NF-κB and YY1 in the regulation of Fas expression was corroborated by the use of Ramos cells with a dominant-active inhibitor of NF-κB (Ramos IκB-estrogen receptor (ER) mutant) and by silencing YY1 with YY1 siRNA, respectively. Further, the role of rituximab-mediated inhibition of the p38 MAPK/NF-κB/YY1 pathway in the regulation of Fas and sensitization to CH-11-induced apoptosis was validated by the use of specific chemical inhibitors of this pathway and which mimicked rituximab-mediated effects. These findings provide a novel mechanism of rituximab-mediated activity by sensitizing NHL cells to Fas-induced apoptosis.

Expression of X-Linked Inhibitor of Apoptosis Protein Is a Strong Predictor of Human Prostate Cancer Recurrence

Purpose: The X-linked inhibitor of apoptosis protein (XIAP) is associated with cell survival by blocking caspase-mediated apoptosis. We examined the expression patterns of XIAP with regard to human prostate cancer, predicting that XIAP status may predict cancer recurrence and/or clinical outcome. Experimental Design: Immunohistochemistry was done on tissue microarrays constructed from 226 primary prostate cancer specimen. The protein expression distribution was examined across the spectrum of epithelial tissues and its association with standard clinicopathologic covariates and tumor recurrence was examined in 192 outcome-informative patients. Results: The mean XIAP expression was significantly higher in prostate cancer compared with prostatic intraepithelial neoplasia (PIN), normal, and benign prostatic hyperplasia. We observed that XIAP is an independent predictor of tumor recurrence in multivariate Cox proportional hazards analysis in all patients as well as after substratifying by Gleason score. Interestingly, patients with high XIAP levels had a much lower probability of tumor recurrence than those with lower XIAP expression. Even patients with high-grade tumors who had higher XIAP levels had a lower risk of recurrence compared with any patient whose tumors express lower XIAP. Conclusions: XIAP is expressed at higher levels in prostate cancers compared with matched normal tissues. High XIAP expression is strongly associated with a reduced risk of tumor recurrence and is not directly associated with Gleason score, tumor stage, capsular involvement, or preoperative prostate-specific antigen status, suggesting that it is a novel prognosticator and a potential target for prostate cancer diagnosis and therapy. Significantly, these findings provide important and extensive validation of previous results.

Hypoxia Inducible Factor promotes murine allergic airway inflammation and is increased in asthma and rhinitis

To cite this article: Huerta‐Yepez S, Baay‐Guzman GJ, Bebenek IG, Hernandez‐Pando R, Vega MI, Chi L, Riedl M, Diaz‐Sanchez D, Kleerup E, Tashkin DP, Gonzalez FJ, Bonavida B, Zeidler M, Hankinson O. Hypoxia Inducible Factor promotes murine allergic airway inflammation and is increased in asthma and rhinitis. Allergy 2011; 66: 909–918.

Nitric oxide sensitizes tumor cells to TRAIL-induced apoptosis via inhibition of the DR5 transcription repressor Yin Yang 1

Treatment of TRAIL-resistant tumor cells with the nitric oxide donor DETANONOate sensitizes the tumor cells to TRAIL-induced apoptosis concomitantly with DR5 upregulation. The mechanism of sensitization was examined based on the hypothesis that DETANONOate inhibits a transcription repressor Yin Yang 1 (YY1) that negatively regulates DR5 transcription. Treatment of the prostate carcinoma cell lines with DETANONOate inhibited both NF-kappaB and YY1 DNA-binding activities concomitantly with upregulation of DR5 expression. The direct role of YY1 in the regulation of TRAIL resistance was demonstrated in cells treated with YY1 siRNA resulting in TRAIL-induced apoptosis. The role of YY1 in the transcriptional regulation of DR5 was examined in cells treated with a DR5 luciferase reporter system (pDR5) and two constructs, namely, the pDR5/-605 construct with a deletion of the putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of the YY1 DNA-binding site. A significant (3-fold) augmentation of luciferase activity over baseline transfection with pDR5 was observed in cells transfected with the modified constructs. ChIP analysis corroborated the YY1 binding to the DR5 promoter. In vivo, tissues from nude mice bearing the PC-3 xenograft and treated with DETANONOate showed inhibition of YY1 and upregulation of DR5. The present findings demonstrate that YY1 negatively regulates DR5 transcription and expression and these correlated with resistance to TRAIL-induced apoptosis. DETANONOate inhibits both NF-kappaB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis.

Mechanisms of nitric oxide-mediated inhibition of EMT in cancer: Inhibition of the metastasis-inducer Snail and induction of the metastasis-suppressor RKIP

The role of nitric oxide (NO) in cancer has been controversial and is based on the levels of NO and the responsiveness of the tumor type. It remains unclear whether NO can inhibit the epithelial to mesenchymal transition (EMT) in cancer cells. EMT induction is mediated, in part, by the constitutive activation of the metastasis-inducer transcription factor, Snail and EMT can be inhibited by the metastasis-suppressor Raf-1 kinase inhibitor protein (RKIP) and E-cadherin. Snail is transcriptionally regulated by NF-κB and in turn, Snail represses RKIP transcription. Hence, we hypothesized that high levels of NO, that inhibit NF-κB activity, may also inhibit Snail and induce RKIP and leading to inhibition of EMT. We show that treatment of human prostate metastatic cell lines with the NO donor, DETANONOate, inhibits EMT and reverses both the mesenchymal phenotype and the cell invasive properties. Further, treatment with DETANONOate inhibits Snail expression and DNA-binding activity in parallel with the upregulation of RKIP and E-cadherin protein levels. The pivotal roles of Snail inhibition and RKIP induction in DETANONOate-mediated inhibition of EMT were corroborated by both Snail silencing by siRNA and by ectopic expression of RKIP. The in vitro findings were validated in vivo in mice bearing PC-3 xenografts and treated with DETANONOate. The present findings show, for the first time, the novel role of high subtoxic concentrations of NO in the inhibition of EMT. Thus, NO donors may exert therapeutic activities in the reversal of EMT and metastasis.

Modulation of mitochondrial functions by the indirect antioxidant sulforaphane: a seemingly contradictory dual role and an integrative hypothesis.

The chemotherapeutic isothiocyanate sulforaphane (SFN) was early linked to anticarcinogenic and antiproliferative activities. Soon after, this compound, derived from cruciferous vegetables, became an excellent and useful trial for anti-cancer research in experimental models including growth tumor, metastasis, and angiogenesis. Many subsequent reports showed modifications in mitochondrial signaling, functionality, and integrity induced by SFN. When cytoprotective effects were found in toxic and ischemic insult models, seemingly contradictory behaviors of SFN were discovered: SFN was inducing deleterious changes in cancer cell mitochondria that eventually would carry the cell to death via apoptosis and also was protecting noncancer cell mitochondria against oxidative challenge, which prevented cell death. In both cases, SFN exhibited effects on mitochondrial redox balance and phase II enzyme expression, mitochondrial membrane potential, expression of the family of B cell lymphoma 2 homologs, regulation of proapoptotic proteins released from mitochondria, activation/inactivation of caspases, mitochondrial respiratory complex activities, oxygen consumption and bioenergetics, mitochondrial permeability transition pore opening, and modulation of some kinase pathways. With the ultimate findings related to the induction of mitochondrial biogenesis by SFN, it could be considered that SFN has effects on mitochondrial dynamics that explain some divergent points. In this review, we list the reports involving effects on mitochondrial modulation by SFN in anti-cancer models as well as in cytoprotective models against oxidative damage. We also attempt to integrate the data into a mechanism explaining the various effects of SFN on mitochondrial function in only one concept, taking into account mitochondrial biogenesis and dynamics and making a comparison with the theory of reactive oxygen species threshold of cell death. Our interest is to achieve a complete view of cancer and protective therapies based on SFN that can be extended to other chemotherapeutic compounds with similar characteristics. The work needed to test this hypothesis is quite extensive.

In vitro and in vivo sensitization of SW620 metastatic colon cancer cells to CDDP-induced apoptosis by the nitric oxide donor DETANONOate: Involvement of AIF

Tumor cells develop mechanisms that dysregulate apoptotic pathways resulting in resistance to cytotoxic stimuli. Primary SW480 and metastatic SW620 colon cancer cells are resistant to CDDP-induced apoptosis. Apoptosis-inducing factor (AIF) was significantly downregulated in SW620 compared to SW480 cells; while apoptotic mediators such as Bax, Bcl-2, and Bcl(XL) were not altered in these cell lines. Examination of tumor tissues from patients with colon cancer demonstrated a significant downregulation of AIF in patients with advanced disease. The role of AIF expression in resistance was examined. Several lines of evidence suggest the involvement of AIF expression level in the sensitivity of SW620 to CDDP-induced apoptosis: (1) sensitization of SW620 by the NO donor DETANONOate to CDDP-induced apoptosis correlated with the induction of AIF as assessed by RT-PCR and Western blot analysis, (2) treatment of SW620 cells with siRNA AIF, but not with control siRNAs, inhibited DETANONOate-induced sensitization to CDDP apoptosis, (3) sensitization by DETANONOate observed in vitro was corroborated in vivo in nude mice bearing SW620 tumor xenografts and treated with the combination of DETANONOate and CDDP, and (4) tumor tissues derived from the SW620 xenografts revealed significant upregulation of AIF and increased apoptosis by DETANONOate and CDDP combination treatment. Altogether, these findings underscore the potential therapeutic application of NO donors and subtoxic chemotherapeutic drugs in the treatment of advanced colon cancer resistant to conventional chemotherapeutic agents.