Characterisation and manipulation of docetaxel resistant prostate cancer cell lines.

BACKGROUND: There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. RESULTS: The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. CONCLUSION: This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation.

BACKGROUND: Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. METHODS: cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. RESULTS: PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. CONCLUSION: Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

Neonatal encephalopathy is associated with altered perinatal systemic neutrophil apoptosis.

Systemic hypoxia-ischemia at birth may alter the neonatal neutrophil phenotype. In this study, we evaluated alterations in perinatal neutrophil phenotype following systemic hypoxia-ischemia compared with normal controls. Neutrophils from adults (n = 15), normal newborns (n = 20), newborns requiring resuscitation at birth (n = 17), and their respective maternal samples were incubated alone or with lipopolysaccharide (LPS). Surface receptor CD11b (neutrophil activation) and the percentage apoptosis (persistence of inflammatory response) were assessed using flow cytometry. Neutrophil apoptosis was decreased in neonates requiring resuscitation at birth and was further exaggerated in infants who developed mild neurological signs. All infants who required resuscitation were LPS hyporesponsive irrespective of neurological findings. Newborns with severe neurological signs had increased apoptosis and decreased CD11b. Maternal neutrophils were LPS hyporesponsive only if their infants had moderate/severe neurological signs. Infants with mild encephalopathy may display a predominantly proinflammatory neutrophil response with a persistent inflammatory response, whereas those with moderate/severe encephalopathy have a tendency toward immunosuppression.

Effects of heat shock and hypoxia on neonatal neutrophil lipopolysaccharide responses: altered apoptosis, Toll-like receptor-4 and CD11b expression compared with adults.

BACKGROUND: Dysfunctional inflammatory responses have been implicated in several neonatal inflammatory disorders following infection and hypoxia. OBJECTIVES: We aimed to study the effects of in vitro hypoxia and heat shock (HS) on normal adult and newborn neutrophil migration (CD11b) and persistence (apoptosis) following lipopolysaccharide (LPS) stimulation. METHODS: The mechanism for altered LPS responses was assessed at the level of the LPS signalling receptors, Toll-like receptor-4 (TLR-4), TLR-2 and CD14 expression in normal neonates and adults. RESULTS: In adults, although hypoxia delayed neutrophil apoptosis, LPS enhanced this response. In contrast, HS (42 degrees C) increased adult apoptotic rates and abrogated the LPS responses. Both hypoxia and HS prevented the LPS-induced increase in adult CD11b although it was unaltered in neonates. Adult TLR-4 neutrophil expression was increased by LPS and hypoxia, and decreased in HS, possibly explaining their variable LPS responsiveness. In contrast, neonatal neutrophils were LPS hyporesponsive which may be mediated by failure of TLR-4 upregulation with LPS. CONCLUSIONS: Neonates do not have increased LPS responsiveness in hypoxia or heat shock in vitro, which may prevent hyperinflammation and thereby minimise tissue damage in inflammation or infection.

TGF-beta1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation.

Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-beta(1) promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-beta(1) stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-beta(1) with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-beta(1) caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-beta(1) treatment. EGF inhibited apoptosis and facilitated TGF-beta(1) induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-beta(1)-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-beta(1) induces EMT independently of its proapoptotic effects. TGF-beta(1) and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type.

Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor have differential effects on neonatal and adult neutrophil survival and function.

Neutropenia is a common sequela of neonatal sepsis. Recent clinical trials have shown the beneficial effects of colony-stimulating factors (CSFs) on outcome in this group, but the exact mechanism remains unknown. Neonates and mothers who were at high-risk for infection were recruited for cord blood sampling in a university tertiary referral maternity hospital. Neonatal and adult neutrophils were evaluated for their ability to combat bacterial infection by examining their functional activity (CD11b and reactive oxygen intermediates) and their persistence at inflammatory sites (apoptosis). The mechanism for altered apoptotic responses was assessed by caspase activation assays, X chromosome-linked inhibitor of apoptosis protein expression, and cytosolic cytochrome c release. Although granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly delayed neutrophil apoptosis in normal adults, only G-CSF had a similar effect in normal neonates. Neutrophils from neonates who are at high risk for infection are unresponsive to the antiapoptotic effects of G-CSF or GM-CSF, unlike maternal neutrophils, which have delayed apoptosis in response to GM-CSF. However, CD11b expression and reactive oxygen intermediate production were significantly increased in normal neonatal neutrophils that were incubated with GM-CSF versus controls but not G-CSF or lipopolysaccharide. Decreased cytosolic cytochrome c release and caspases 3 and 9 activity are associated with the CSF-mediated delay in apoptosis in adults but not in newborns. The antiapoptotic X chromosome-linked inhibitor of apoptosis protein is up-regulated in neonates compared with adults and may mediate their differential spontaneous apoptosis. These results have important implications for the use of CSFs in neonatal sepsis, as responses differ from those seen in adults. Further delineation of neonatal neutrophil responses to CSFs may improve their therapeutic potential.

Labor promotes neonatal neutrophil survival and lipopolysaccharide responsiveness.

Labor is a mild proinflammatory state that is associated with fetal leukocytosis. Elective cesarean section has been linked with increased neonatal morbidity, which may be partially immune mediated. We hypothesized that labor may alter neutrophil phenotype and thereby decrease neonatal complications. We characterized neutrophil function and survival in normal neonates after either uncomplicated vaginal delivery (VD) or elective cesarean section (CS) without labor. Spontaneous neutrophil apoptosis is delayed in cord blood neutrophils of neonates after normal labor (VD) compared with CS, as assessed by propidium iodide DNA incorporation using flow cytometry. This demonstrates their ability to maintain an inflammatory response. CD11b expression on neonatal neutrophils after CS is decreased, providing further evidence of altered activation or priming. Lipopolysaccharide responsiveness, characterized by CD11b and apoptosis, is similar in VD and adults, but CS-derived neutrophils are unresponsive. Baseline TLR-4 levels are elevated in CS in contrast to the other groups, although expression is not up-regulated by lipopolysaccharide co-incubation. Neonatal neutrophil survival and function are altered by labor and may increase antibacterial function and neutrophilia. This suggests that labor of any duration may be immunologically beneficial to the normal term neonate.

Aspirin preserves neutrophil apoptosis after cardiopulmonary bypass.

The aim of this study was to test the hypothesis that ongoing aspirin therapy preserves neutrophil apoptosis after cardiac surgery with cardiopulmonary bypass (CPB) by a cyclooxygenase mechanism. Twenty patients undergoing coronary revascularization with CPB were enrolled in a prospective cohort study. Patients who had continued taking 300 mg of aspirin until the day before surgery (n = 10) were compared with 10 patients not taking aspirin or who had discontinued it more than 5 days before surgery. Neutrophils were isolated from arterial blood before and 6 h after surgery and apoptosis was measured after 24 h in culture using flow cytometry. Serum was collected and assessed for IL-6, IL-8 and PGE2 by enzyme-linked immunoabsorbant assay. Patients were followed for clinical indices of sepsis for 7 days postoperatively. Spontaneous rates of neutrophil apoptosis were significantly reduced in postoperative compared with preoperative samples. There was no difference between aspirin and control preoperative neutrophil apoptosis rates (23.0% +/- 11.3% vs. 23.0% +/- 20.7%, P = 0.99). Postoperative neutrophil apoptosis was delayed in control patients (3.6% +/- 1.2% apoptosis), but this was significantly (P = 0.045) reversed in the aspirin-treated group (7.2% +/- 5.1% apoptosis). There were lower postoperative PGE2 levels in the aspirin group (136 +/- 69 pg/mL vs. 372 +/- 210 pg/mL, P = 0.04). There was no difference in clinical indices of sepsis. We conclude that the delay in postoperative neutrophil apoptosis is significantly preserved in patients taking 300 mg of aspirin on the day before surgery. This was associated with greater inhibition of PGE2, consistent with the hypothesis that aspirin exerts its effect on apoptosis after CPB via a cyclooxygenase-mediated mechanism.

Gene expression profile of inflammatory neutrophils: alterations in the inhibitors of apoptosis proteins during spontaneous and delayed apoptosis.

Inflammatory mediators delay neutrophil apoptosis, which contributes to the persistence of inflammation. The mechanisms responsible for this delay and resistance to Fas antibody-induced apoptosis are unknown but are dependent on protein synthesis. These proteins have been shown to inhibit caspase activity central to the induction of apoptosis. The inhibitors of apoptosis proteins have been shown to inhibit caspase activity and prevent apoptosis in a number of cellular systems. We hypothesize that the regulation of neutrophil apoptosis is dependent on the expression of the IAPs. c-IAP-1, c-IAP-2, and XIAP are expressed in the neutrophil at both the mRNA and protein level, but their relative protein expression is low compared with other cell types. The in vitro aging of human neutrophils results in their induction of apoptosis, which is associated with the loss of c-IAP-1 expression. The pancaspase inhibitor (zVAD-FMK) and LPS, which delay spontaneous apoptosis, also prevented this loss of c-IAP-1. Gene chip microarrays have shown that LPS increases c-IAP-1 and c-IAP-2 mRNA expression in neutrophils. However, this does not correspond to an increase in protein. Neutrophils from septic patients with delayed apoptosis show an increase in XIAP, with no change in cIAP-1 or cIAP-2 mRNA, demonstrating that different mechanisms contribute to the delay in neutrophil apoptosis. This study demonstrates that the loss of IAP expression may facilitate the induction of neutrophil apoptosis, and preventing this loss of IAP expression may represent a more significant contribution to delayed apoptosis rather than an increase in their expression.

Labor induces a maternal inflammatory response syndrome.

OBJECTIVE: We studied the effect of labor on maternal neutrophil phenotype. STUDY DESIGN: Neutrophil apoptosis with inflammatory cytokines and the expression of CD11b, CD34 and toll-like receptor 4 (TLR4) were assessed with flow cytometry in women at uncomplicated vaginal delivery (VD), and elective cesarean section (ElCS) without labor, emergency cesarean section with (EmCSL+) or without (EmCSL-) labor. RESULTS: Spontaneous neutrophil apoptosis is delayed in maternal neutrophils after VD, EmCSL+ or EmCSL- versus ElCS. In all groups lipopolysaccharide delayed apoptosis and increased CD11b expression. Elevated TLR4 expression in ElCS was associated with lipopolysaccharide responsiveness. CD34 was diminished in VD, indicating increased cell maturity. CONCLUSION: Normal labor primes the neutrophil and may enhance antibacterial function by inducing a mild maternal inflammatory response syndrome. Delayed neutrophil apoptosis may promote the neutrophilia seen in women after VD. We suggest that labor of any duration may be immunologically beneficial to the parturient.

Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone.

Women are conferred with greater immunologic and survival benefits compared to men. Female sex steroids contribute to this sexual dimorphism. Furthermore, during human pregnancy when female sex hormones are elevated, neutrophil apoptosis is delayed. This study examines the specific effects of estradiol and progesterone on neutrophil apoptosis and function in healthy adult men and women. We also examined the contribution of these hormones to the persistence and resolution of an inflammatory response. Spontaneous apoptosis was significantly decreased in women compared with men. Physiologic doses of estradiol and progesterone caused a further delay in spontaneous apoptosis in both men and women but did not diminish Fas antibody-induced apoptosis. The delay in apoptosis was mediated at the level of the mitochondria with decreased release of cytochrome c, which may alter caspase cleavage and activity. There were no associated alterations in neutrophil CD11b, but production of reactive oxygen intermediates (ROIs) in women was increased. Thus, female sex hormones mediate delayed neutrophil apoptosis in both sexes and enhance female intracellular production of ROIs. Modulating hormonal responses may be an effective therapeutic tool in combating inflammatory diseases.

The apoptosome pathway to caspase activation in primary human neutrophils exhibits dramatically reduced requirements for cytochrome C.

Caspase activation is a central event in numerous forms of apoptosis and results in the proteolytic degradation of multiple substrate proteins that contribute to the apoptotic phenotype. An important route to caspase activation proceeds via assembly of the "apoptosome" as a result of the cell stress-associated release of mitochondrial cytochrome c. Previous studies have shown that primary neutrophils are largely incapable of mitochondrial respiration, suggesting that these cells either lack functional mitochondria or possess a defective respiratory chain. This prompted us to examine whether neutrophils retain an intact cytochrome c/apoptotic protease-activating factor 1 (Apaf-1) pathway to caspase activation and apoptosis. We show that primary human neutrophils contain barely detectable levels of cytochrome c as well as other mitochondrial proteins. Surprisingly, neutrophil cell-free extracts readily supported Apaf-1-dependent caspase activation, suggesting that these cells may assemble cytochrome c-independent apoptosomes. However, further analysis revealed that the trace amount of cytochrome c present in neutrophils is both necessary and sufficient for Apaf-1-dependent caspase activation in these cells. Thus, neutrophils have a lowered threshold requirement for cytochrome c in the Apaf-1-dependent cell death pathway. These observations suggest that neutrophils retain cytochrome c for the purpose of assembling functional apoptosomes rather than for oxidative phosphorylation.

Endothelin in unilateral ureteral obstruction: vascular and cellular effects.

PURPOSE: Unilateral ureteral obstruction results in decreased blood flow and tissue loss in the obstructed kidney. This condition is compensated by increased perfusion and trophic changes in the contralateral kidney. Vascular mediators' effects are central to these changes and of these mediators endothelin is the most potent vasoconstrictor known. We explored the role of endothelin and the effects of endothelin receptor blockade in unilateral ureteral obstruction. MATERIALS AND METHODS: Rats were subjected to unilateral ureteral obstruction for 24 hours. Endothelin-1 mRNA expression was determined in kidney extracts from control, obstructed and contralateral (nonobstructed) kidneys. Cortical and medullary blood flow was determined in control and obstructed kidneys, and after endothelin receptor blockade with bosentan. Apoptotic rates were determined in control and obstructed kidneys after treatment with bosentan using the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end technique. RESULTS After 24 hours of unilateral ureteral obstruction endothelin-1 mRNA expression was increased in the obstructed kidney and decreased in the contralateral kidney. Obstruction was associated with a decrease in renal blood flow, which was reversed by endothelin receptor blockade. Unilateral ureteral obstruction also increased apoptosis, which was blocked by endothelin inhibition. CONCLUSIONS: Endothelin expression increases in the obstructed kidney. Inhibition of its action protects against vascular and cellular changes. Decreased endothelin expression in the contralateral kidney may facilitate trophic changes and compensatory increased blood flow.

Androgen-mediated resistance to apoptosis.

BACKGROUND: Previous studies demonstrate that androgen is capable of exerting a protective effect in the androgen-sensitive human prostate cancer cell line LNCaP. Limited studies, however, have addressed the underlying mechanisms involved, in particular the effects of androgen on both pro- and anti-apoptotic gene expression. METHODS: We investigated the effects of androgen on apoptotic sensitivity and the expression of the caspases and specific members of the Bcl-2 family in the LNCaP cell line. The effects of androgen on NF-kappaB activation were also investigated by using a gel mobility shift assay. RESULTS: 5alpha-Dihydrotestosterone (5-alphaDHT) conferred resistance to radiation (5 Gy) and etoposide-induced apoptosis in the LNCaP cell line. This finding was associated with a time-dependent decrease in the expression of the caspases and pro-apoptotic Bcl-2 family members. 5-alphaDHT did not confer protection against apoptosis in the LNCaP line transfected with the IkappaB super repressor of NF-kappaB, nor in the androgen insensitive PC-3 and DU-145 cell lines. CONCLUSION: The ability of 5-alphaDHT to raise the apoptotic threshold in the LNCaP cell line by altering specific pro-apoptotic gene expression suggests that androgen may serve as a general survival signal against diverse pathways that ultimately signal for apoptosis. We hypothesize that NF-kappaB serves as an important mediator in androgen survival signaling.

Cytoprotective effects of nitrates in a cellular model of hydronephrosis.

BACKGROUND: The earliest insult to the kidney following the onset of ureteral obstruction is a marked elevation in collecting system pressure. This imparts a mechanical stress that is transmitted directly from the collecting system to the kidney substance. Renal tubular injury is the principal functional and histological change encountered, with glomerular changes being less marked and occurring later. Nitric oxide (NO) has been shown to protect against renal injury in UO, but its mode of action has not been clearly defined. METHODS: MDCK (canine) and HK-2 (human) renal tubular cells were grown under control conditions or subjected to mechanical strain for periods of 24 and 48 hours. Cells were studied treated with or without Fas-antibody, etoposide or diethyl maleate (DEM) alone or in combination with NG-monomethyl l-arginine (L-NMMA), sodium nitroprusside (SNP) or l-arginine. Cell proliferation and apoptosis was determined using propidium iodide DNA staining. NO production and inducible NO synthase (iNOS) expression were measured by the Griess reaction and Western blotting, respectively. RESULTS: Cells subjected to mechanical strain displayed a decrease in the proportion of cells undergoing cell division. They also showed an increased susceptibility to apoptosis. Associated with this was a decrease in Bcl-2 expression. An increase in iNOS expression was seen in cells subjected to mechanical strain, but no increase in NO production. The cellular effects of mechanical strain were reversed by SNP and l-arginine. CONCLUSIONS: Culture of renal tubule cells in an environment of mechanical strain results in an imbalance in homeostasis and a net cell loss. This can be reversed by the administration of an NO donor or precursor.

Inhibitors of apoptosis proteins in prostate cancer cell lines.

BACKGROUND: The caspases are the central executioners of apoptosis. The inhibitors of apoptosis proteins (IAPs) are a family of recently described caspase inhibitors. We hypothesised that tumor resistance to apoptosis could be due in part to IAP expression. METHODS: The expression of NAIP, cIAP-1, cIAP-2, XIAP, and survivin was investigated in the prostate cancer cell lines LNCaP, PC3, and DU145. RNase protection assays and Western blotting were used to assess RNA and protein expression. Apoptotic susceptibility was determined using etoposide and assessed by propidium iodide (PI) DNA incorporation using flow cytometry. RESULTS: DU145 and PC3 cells were more resistant to apoptosis than LNCaP cells. All the IAPs were identified in the cell lines with variation in IAP expression between different cell types. Immunohistochemistry demonstrated cIAP-1 expression in PC3 cells was nuclear, while the expression of cIAP-2 and XIAP was perinuclear. Growing LNCaP cells in charcoal-stripped or androgen-supplemented medium resulted in no alteration in IAP expression. CONCLUSIONS: This study characterises the expression of IAP in three of the most commonly used prostate cancer cells. IAP may make an important contribution to apoptotic resistance in patients with prostate cancer.