No abstract

Previous studies documented the ability of quinazoline-based a1-adrenoceptor antagonists to induce apoptosis in prostate cancer cells via an a1-adrenoceptor-independent mechanism. In this study we investigated the molecular events initiating this apoptotic effect. Since transforming growth factor-b1 (TGF-b1) mediates prostate epithelial cell apoptosis, we hypothesised that the activation of the TGF-b1 pathway underlies the quinazoline-based apoptotic effect in prostate cancer cells. Treatment of the androgenindependent human prostate cancer cells PC-3 with doxazosin resulted in a strong caspase-3 activation within 24 h, whereas tamsulosin, a sulphonamide-based a1-adrenoceptor antagonist, had no significant apoptotic effect against prostate cancer cells. To identify the molecular components involved in this quinazoline-mediated apoptosis, cDNA microarray analysis of PC-3 prostate cancer cells treated with doxazosin (3 h) was performed. Induced expression of several genes was observed including p21 WAF-1 and IkBa (inhibitor of NF-kB alpha). Relative quantitative reverse transcription -polymerase chain reaction analysis revealed induction of several TGF-b1 signalling effectors: Induction of mRNA for Smad4 and the TGF-b1-regulated apoptosis-inducing transcription factor TGF-b1-inducible early gene (TIEG1) was detected within the first 6 h of doxazosin treatment. Upregulation of IkBa at both the mRNA and protein level was also detected after 6 h of treatment. Furthermore, doxazosin resulted in a considerable elevation in Smad4 and TIEG protein expression (6 h). A 'latent' increase in TGF-b mRNA expression was detected after 48 h of treatment. These findings suggest that the quinazoline-based doxazosin mediates prostate cancer apoptosis by initially inducing the expression of TGF-b1 signalling effectors and subsequently IkBa. The present study provides an initial insight into the molecular targets of the apoptotic action of quinazolines against prostate cancer cells.

Removal of the synaptic targets of olfactory receptor neurons by olfactory bulb ablation results in apoptosis of olfactory receptor neurons and up-regulation of proliferation of their progenitors. This study focuses on the expression of the neuropoietic cytokines leukemia inhibitory factor (LIF) and its receptor (LIFR) and interleukin 6 (IL-6) and its receptor (IL-6R) in intercellular signaling pathways in the olfactory mucosa after target ablation. Olfactory bulbectomy (OBX) resulted in several transient, early-onset, temporally integrated events that were detected immunohistochemically. Macrophages infiltrated the olfactory epithelium (OE) by 16 hours post-OBX. LIF expression was up-regulated transiently at 2 days post-OBX, when up-regulated expression of LIFR also was detected on globose basal cells (GBCs), a subpopulation of which are immediate progenitors of olfactory receptor neurons. GBC proliferation peaked at 3--4 days post-OBX. In the olfactory nerve (ON), LIF-positive and IL-6-positive macrophage infiltration was followed by the transient up-regulation of expression of LIFR, IL-6, and IL-6R in ensheathing cells by 3 days post-OBX. The mRNAs for LIF/LIFR, IL-6/IL-6R, and their common signal-transduction molecule, gp130, in olfactory-nasal mucosa from control mice and from 3-day post-OBX mice were detected with reverse transcriptase-polymerase chain reaction (RT-PCR). Analysis of Northern blot and relative quantitative RT-PCR demonstrated similar temporal patterns of changes in relative mRNA levels for both LIF and IL-6, which were up-regulated by 16 hours post-OBX and peaked at 2--3 days post-OBX. These data indicate that LIF from infiltrating macrophages acts as a mitogen for GBCs and that LIF from infiltrating macrophages and IL-6 from infiltrating macrophages and ensheathing cells act as repair factors in the ON.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.