Early effects of pharmacological androgen deprivation in human prostate cancer.

OBJECTIVES: To assess the early histological effects of pharmacological androgen deprivation (AD), which have been assessed only over longer periods, as surgical castration leads rapidly to diminished cell proliferation and enhanced cell death within the prostate. PATIENTS AND METHODS: With Institutional Review Board approval, 35 patients were randomly assigned (seven in each group) to receive 0, 7, 14, 21 and 28 days of AD (flutamide, 250 mg orally three times/day, and one injection with leuprolide acetate 7.5 mg) before radical prostatectomy. The surgical specimens were assessed by conventional histology and immunohistochemistry, while macroarray analysis and quantitative real-time polymerase chain reaction (QRT-PCR) were used to examine gene expression. RESULTS: There were morphological changes within the prostatic tissues as early as 7 days after initiating AD, similar to the response to castration. There was tumour cell vacuolization indicating cellular injury, glandular atrophy and mononuclear cell infiltration as prominent and progressive effects but, by contrast with castration studies, there were no changes in epithelial proliferation or apoptosis. Macroarray analysis, validated by QRT-PCR and immunohistochemistry, showed up-regulation of numerous and potentially counter-effective genes involved in the cell cycle and apoptosis. CONCLUSIONS: Pharmacological AD induces significant involution within prostatic tissues over 7-28 days, but allows the persistence of some viable tumour cells capable of proliferation.

Knock down of p53 levels in human keratinocytes increases susceptibility to type I and type II interferon-induced apoptosis mediated by a TRAIL dependent pathway.

BACKGROUND: Keratinocytes (KCs) in healthy skin only undergo death following differentiation to produce stratum corneum. By contrast, in inflammatory pathological conditions featuring type I (IFN-alpha) and type II (IFN-gamma) interferons KCs undergo premature apoptosis. OBJECTIVE: To define apoptotic susceptibility of KCs, response to interferons was examined. Since molecular cross-talk occurs between interferons and p53, potential mechanistic roles for p53 in KC apoptosis were investigated. METHODS: Knock down of p53 was performed, and apoptotic response to addition of interferons was assessed using FACS and by staining for activated caspase 3 and TUNEL. Elucidation of death pathway was accomplished by using a dominant negative death receptor construct and a neutralizing TRAIL antibody. RESULTS: Reduction in p53 levels in KCs by siRNA treatment enhanced, rather than reduced, apoptotic responses to IFN-alpha plus IFN-gamma. In an immortalized human KC cell line (HaCaT cells with both p53 alleles mutated) enhanced apoptotic susceptibility to interferon exposure was also observed. The mechanism for this enhanced apoptosis involved induction of TRAIL and its interaction with death receptors, as blocking the death receptor pathway using dominant negative FADD, or by addition of neutralizing antibody against TRAIL, reduced the apoptotic response to IFN-alpha and IFN-gamma. CONCLUSION: These results indicate IFN-alpha plus IFN-gamma triggers apoptosis independent of p53 in HaCaT cells, and also demonstrate an unexpected survival role for p53 in human KCs as regards apoptotic responsiveness to cytokines such as IFN-alpha and IFN-gamma involving activation of TRAIL-related death receptors. Strategies enhancing p53 regulated survival proteins in KCs may be of therapeutic benefit in skin disorders characterized by activated immunocytes triggering premature KC apoptosis.

Defining the caspase-containing apoptotic machinery contributing to cornification in human epidermal equivalents.

Whether terminal differentiation/stratum corneum formation of keratinocytes (KCs) represents a form of programmed cell death, utilizing mediators of classical apoptosis, is unclear. Apoptosis, an evolutionarily conserved death process, is comprised of extrinsic and intrinsic pathways, which converge using caspase 3. To define upstream and downstream caspases involved in terminal differentiation, we utilized human epidermal equivalents (EEs). Using submerged cultures comprised of human KCs, EEs were sequentially analyzed before and after being raised to an air/liquid (A/L) interface at 3-24 h intervals. At each time point, EEs were analyzed morphologically and for specific enzyme activity to distinguish different initiator (caspases 1, 2, 8, 9) and effector caspases (3, 6, 7). Terminal differentiation began at 6-8 h, as defined by stratum corneum with loricirin expression and completed at 18-24 h producing an epidermis resembling normal skin. Enzyme activity for caspases 1, 2, 3, 6, 7, 8, and 9 (but not 4, 5) was enhanced (>two-fold nmol/mg/h) at 3-6 h compared with submerged cultures. Processing of caspase 14 occurred at 18 h, and cleaved caspase 14 was increased at 24 h. Activated caspase 3-positive and terminal deoxynucleotidyl transferase-mediated nick end labeling-positive KCs were identified in EEs at 3-6 h corresponding to initiation sites of terminal differentiation. Addition of caspase inhibitors reduced levels of involucrin and loricrin in EEs raised to an A/L interface. We conclude caspases function as important death effectors strategically positioned at intersection of intrinsic and extrinsic pathways in KCs undergoing stratum corneum formation.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells.

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Knockdown of p53 levels in human keratinocytes accelerates Mcl-1 and Bcl-x(L) reduction thereby enhancing UV-light induced apoptosis.

Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50-80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-x(L) antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-x(L) reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-x(L) levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas.

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Tumor suppressor maspin is up-regulated during keratinocyte senescence, exerting a paracrine antiangiogenic activity.

Cell senescence is a physiological program of terminal growth arrest, which is believed to play an important role in cancer prevention. Senescent cells secrete multiple growth-regulatory proteins, some of which can affect tumor growth, survival, invasion, or angiogenesis. Changes in expression of different senescence-associated genes were analyzed in cultured human skin keratinocytes (KCs) that underwent replicative senescence or confluence-induced accelerated senescence. Senescent KC cultures showed a strong increase in mRNA and protein expression of maspin, a member of serine protease inhibitor family and an epithelial cell tumor suppressor with anti-invasive and antiangiogenic activities. Immunohistochemical analysis of 14 normal human skin samples (age range from 3 months to 84 years) showed that maspin is expressed by KCs in vivo and that the extent and intensity of maspin expression in the skin is significantly (P = 0.01) correlated with chronological age. Antiangiogenic activity of maspin secreted by senescent KCs was investigated in vitro by testing the effect of conditioned media from different KC cultures on endothelial cell migration in the presence or absence of several angiogenic factors. Media conditioned by senescent cultures (undergoing replicative or accelerated senescence), but not by proliferating KCs, strongly inhibited the stimulation of endothelial cell migration by all of the tested angiogenic factors. Neutralizing antibody against maspin abrogated this effect of conditioned media. These findings indicate that senescent KCs exert a paracrine antiangiogenic activity, and maspin is the principal contributor to this potentially tumor-suppressive effect of cellular senescence.