Apoptosis is induced in Chlamydia trachomatis infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.

Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1

The Wilms’ tumor suppressor protein WT1 is a transcriptional regulator involved in differentiation and the regulation of cell growth. WT1 is subject to alternative splicing, one isoform including a 17–amino acid region that is specific to mammals. The function of this 17–amino acid insertion is not clear, however. Here, we describe a transcriptional activation domain in WT1 that is specific to the WT1 splice isoform that contains the 17–amino acid insertion. We show that the function of this domain in transcriptional activation is dependent on a specific interaction with the prostate apoptosis response factor par4. A mutation in WT1 found in Wilms’ tumor disturbs the interaction with par4 and disrupts the function of the activation domain. Analysis of WT1 derivatives in cells treated to induce par4 expression showed a strong correlation between the transcription function of the WT1 17–amino acid insertion and the ability of WT1 to regulate cell survival and proliferation. Our results provide a molecular mechanism by which alternative splicing of WT1 can regulate cell growth in development and disease.

BRCA1 is an essential mediator of vinorelbine-induced apoptosis in mesothelioma

The rapeutic options for malignant pleural mesothelioma (MPM) are limited despite the increasing incidence globally. The vinca alkaloid vinorelbine exhibits clinical activity; however, to date, treatment optimization has not been achieved using biomarkers. BRCA1 regulates sensitivity to microtubule poisons; however, its role in regulating vinorelbine-induced apoptosis in mesothelioma is unknown. Here we demonstrate that BRCA1 plays an essential role in mediating vinorelbine-induced apoptosis, as evidenced by (1) the strong correlation between vinorelbine sensitivity and BRCA1 expression level; (2) induction of resistance to vinorelbine by BRCA1 using siRNA oligonucleotides; (3) dramatic down-regulation of BRCA1 following selection for vinorelbine resistance; and (4) the re-activation of vinorelbine-induced apoptosis following re-expression of BRCA1 in resistant cells. To determine whether loss of BRCA1 expression in mesothelioma was potentially relevant in vivo, BRCA1 immunohistochemistry was subsequently performed on 144 primary mesothelioma specimens. Loss of BRCA1 protein expression was identified in 38.9% of samples. Together, these data suggest that BRCA1 plays a critical role in mediating apoptosis by vinorelbine in mesothelioma, warranting its clinical evaluation as a predictive biomarker. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chronic immune activation and inflammation as the cause of malignancy

Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers. © 2001 Cancer Research Campaign http://www.bjcancer.com.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

Background Although PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents. Methods We have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2). Results 15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis. Conclusion Troglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis

Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity. However, the mechanism of AGP-induced cancer cell death is unknown. In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells. By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells. TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis. Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis. Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis. The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

The effect of mechanochemical activation upon the intercalation of a high-defect kaolinite with formamide

The effect of mechanochemical activation upon the intercalation of formamide into a high-defect kaolinite has been studied using a combination of X-ray diffraction, thermal analysis, and DRIFT spectroscopy. X-ray diffraction shows that the intensity of the d(001) spacing decreases with grinding time and that the intercalated high-defect kaolinite expands to 10.2 A. The intensity of the peak of the expanded phase of the formamide-intercalated kaolinite decreases with grinding time. Thermal analysis reveals that the evolution temperature of the adsorbed formamide and loss of the inserting molecule increases with increased grinding time. The temperature of the dehydroxylation of the formamide-intercalated high-defect kaolinite decreases from 495 to 470oC with mechanochemical activation. Changes in the surface structure of the mechanochemically activated formamide-intercalated high-defect kaolinite were followed by DRIFT spectroscopy. Fundamentally the intensity of the high-defect kaolinite hydroxyl stretching bands decreases exponentially with grinding time and simultaneously the intensity of the bands attributed to the OH stretching vibrations of water increased. It is proposed that the mechanochemical activation of the high-defect kaolinite caused the conversion of the hydroxyls to water which coordinates the kaolinite surface. Significant changes in the infrared bands assigned to the hydroxyl deformation and amide stretching and bending modes were observed. The intensity decrease of these bands was exponentially related to the grinding time. The position of the amide C&unknown;O vibrational mode was found to be sensitive to grinding time. The effect of mechanochemical activation of the high-defect kaolinite reduces the capacity of the kaolinite to be intercalated with formamide.