15-deoxy-Delta-12,14-prostaglandin J2 induces programmed cell death of breast cancer cells by a pleiotropic mechanism

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been found to induce cell death in a variety of cells. In this regard, we reported recently that 15-deoxy-Delta-(12,14)-prostaglandin J2 (15dPG-J2), a specific ligand of the nuclear receptor PPARgamma, inhibits proliferation and induces cellular differentiation and apoptosis in the breast cancer cell line MCF-7. In addition to PPARgamma activation other proteins, such as NF-kappaB and AP1, have been shown to be targets of 15dPG-J2. However, the mechanism by which 15dPG-J2 triggers cell death is still elusive. Our results demonstrate that 15dPG-J2 initiates breast cancer cell death via a very rapid and severe impairment of mitochondrial function, as revealed by a drop in mitochondrial membrane potential (DeltaPsi(m)), generation of reactive oxygen species (ROS) and a decrease in oxygen consumption. In addition, 15dPG-J2 can also activate an intrinsic apoptotic pathway involving phosphatidyl serine externalization, caspase activation and cytochrome c release. Bcl-2 over-expression and zVADfmk, albeit preventing caspase activation, have no effect on 15dPG-J2-mediated mytochondrial dysfunction and loss of cell viability. In contrast, the addition of radical scavengers or rotenone, which prevent 15dPG-J2-induced ROS production, block the loss of cell viability induced by this prostaglandin. Finally, 15dPG-J2-induced cell death appears to involve disruption of the microtubule cytoskeletal network. Together, these results suggest that PG-J2-induced mitochondrial dysfunction and ROS production inevitably leads to death, with or without caspases.     

Prostaglandin J2 and 15-deoxy-delta12,14-prostaglandin J2 induce proliferation of cyclooxygenase-depleted colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Nonsteroidal anti-inflammatory agents (NSAIDS) inhibit growth of various CRC cell lines by both COX-dependent and COX-independent pathways. To specifically examine the effect of COX and PGs on proliferation in CRC cells, we introduced an antisense COX-2 cDNA construct under the control of a tetracycline (Tc)-inducible promoter into a CRC cell line, HCA-7, Colony 29 (HCA-7) that expresses COX and produces PGs. In the presence of Tc, PG production in COX-depleted cells was reduced 99.8% compared with either uninduced transfectants or parental HCA-7 cells. This decrease in PG production was associated with a concomitant 60% reduction in DNA replication. Subsequently, we examined the effects of various PGs to modulate cell growth in COX-depleted HCA-7 or COX-null HCT-15 cells by quantifying [3H]thymidine incorporation and/or growth in collagen gels. We report that J-series cyclopentenone PGs, particularly PGJ2 and 15-deoxy-delta12,14-PGJ2, induce proliferation of these cells at nanomolar concentrations. Lipids extracted from parental HCA-7 cell conditioned medium stimulated mitogenesis in COX-depleted HCA-7 cells and COX-null HCT-15 cells. Using chromatographic and mass spectrometric approaches, we were able to detect PGJ2 in conditioned medium from parental HCA-7 cells. Taken together, these findings implicate a role for cyclopentenone PGs in CRC cell proliferation.     

Involvement of c-jun N-terminal kinase activation in 15-deoxy-delta12,14-prostaglandin J2-and prostaglandin A1-induced apoptosis in AGS gastric epithelial cells

Cyclopentenone prostaglandins (CyPGs), derivatives of arachidonic acid, have been suggested to exert growth-inhibitory activity through peroxisome proliferator-activated receptor (PPAR)-dependent and -independent mechanisms. Here we examined various eicosanoids for growth inhibition and found that the terminal derivative of prostaglandin (PG) J(2) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), and PGA(1) markedly inhibited the growth and induced apoptosis in AGS gastric carcinoma cells. There were no significant increases in cell death and DNA-fragmentation in the cells with overexpression of PPARalpha or PPARgamma, indicating the possibility that 15d-PGJ(2) and PGA(1) induced apoptosis through PPAR-independent pathway. Moreover, 15d-PGJ(2) and PGA(1) activated the c-jun N-terminal kinase (JNK) and caspase-3 activity in dose- and time-dependent manners. To examine further the role of JNK signaling cascades in apoptosis induced by 15d-PGJ(2) and PGA(1), we transfected dominant-negative (DN) mutants of JNK plasmid into the cells to analyze the apoptotic characteristics of cells overexpressing DN-JNK following exposure to 15d-PGJ(2) and PGA(1). Overexpression of DN-JNK significantly repressed both endogenous JNK and caspase-3 activity, and subsequently decreased apoptosis induced by 15d-PGJ(2) and PGA(1). These results suggested that CyPGs, such as 15d-PGJ(2) and PGA(1), activated JNK signaling pathway, and that JNK activation may be involved in 15d-PGJ(2)- and PGA(1)-induced apoptosis.     

Expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-gamma and levels of prostaglandin E2 and 15-deoxy-delta12,14-prostaglandin J2 in human breast cancer and metastasis

Cyclooxygenase-2 (COX-2) expression and peroxisome proliferator-activated receptor-gamma (PPARgamma) inactivation are linked to increased risk of human breast cancer. The purpose of our study was to examine the relationship between COX-2 (with the resulting prostaglandins E(2), PGE(2)) and PPARgamma (and its natural endogenous ligand 15-Deoxy-Delta(12,14)-prostaglandin J(2), 15d-PGJ(2)) at various stages during the development of human breast cancer and its progression to metastasis. Human breast tissue specimens were collected from normal breasts or from individuals with fibrocystic disease and served as controls (n = 22). Tissues were also collected from uninvolved (n = 25), tumor (n = 25) and lymph node metastasis (n = 15) regions from breast cancer patients. COX-2 and PPARgamma mRNA expression were increased and downregulated, respectively, in tissues from cancer patients compared to controls. Metastatic tissues tended to have higher alterations compared to non-metastatic tissues (p < 0.05). These altered expressions in COX-2 and PPARgamma were paralleled by increases in the tissue levels of PGE(2) and decreases in 15d-PGJ(2). A significant inverse correlation was found between PGE(2) and 15-d-PGJ(2) (r = -0.51, p < 0.05). Significant correlations (p < 0.05) were also obtained between COX-2 and PPARgamma mRNA (inverse, r = -0.72) and between COX-2 and PGE(2) (direct, r = 0.68). Increases in COX-2 mRNA expression and levels of PGE(2) and down-regulation of PPARgamma mRNA expression and 15d-PGJ(2) levels were characterized as predictors of breast cancer risk (p < 0.05). Our results suggest that the altered expression of COX-2 and PPARgamma and the subsequent modulation in the tissue levels of PGE(2) and 15-d-PGJ(2) may influence the development of human breast cancer and its progression to metastasis.     

Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

Background  

The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.     

Role of SNTA1 in Rac1 activation,modulation of ROS generation,and migratory potential of human breast cancer cells

Background:

Alpha-1-syntrophin (SNTA1) has been implicated in the activation of Rac1. However, the underlying mechanism has not yet been explored. Here, we show that a novel complex, involving SNTA1, P66shc, and Grb2 proteins, is involved in Rac1 activation.

Methods:

Co-immunoprecipitation assays were used to show the complex formation, while siRNAs and shRNAs were used to downregulate expression of these proteins. Various Rac1 activation assays and functional assays, such as migration assays, in vitro wound healing assays, cell proliferation assays, and ROS generation assays, were also performed.

Results:

The results showed a significant increase in activation of Rac1 when SNTA1 and P66shc were overexpressed, whereas depletion of SNTA1 and P66shc expression effectively reduced the levels of active Rac1. The results indicated a significant displacement of Sos1 protein from Grb2 when SNTA1 and P66shc are overexpressed in breast cancer cell lines, resulting in Sos1 predominantly forming a complex with Eps8 and E3b1. In addition, the SNTA1/P66shc-mediated Rac1 activation resulted in an increase in reactive oxygen species (ROS) production and migratory potential in human breast cancer cells.

Conclusion:

Together, our results present a possible mechanism of Rac1 activation involving SNTA1 and emphasise its role in ROS generation, cell migration, and acquisition of malignancy.     

Potentiation of tumor formation by topical administration of 15-deoxy-delta12,14-prostaglandin J2 in a model of skin carcinogenesis

The effect of prostaglandins on the development of papillomas has been investigated in mice receiving prostaglandins E2 (PGE2) or the cyclopentenone 15-deoxy-delta(12,14)-PGJ2 (15dPGJ2) topically, using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced tetradecanoylphorbol acetate (TPA)-promoted model of skin carcinogenesis. The presence of 15dPGJ2 during DMBA and TPA treatment inhibited apoptosis and increased the rate, number, size and vascularization of the papillomas, some of them progressing into carcinomas. Moreover, skin sections from mice treated for one week with DMBA and 15dPGJ2 showed a much reduced rate of apoptotic cells, and an enhanced expression of vascular epithelial growth factor when compared with animals receiving DMBA, with or without PGE2. The analysis of molecular events in the MCA3D keratinocyte cell line showed that 15dPGJ2 activated Ras and improved cell viability by inhibiting DMBA-dependent apoptosis. In addition to this, cell adhesion was impaired in MCA3D keratinocytes co-treated with 15dPGJ2 and DMBA, at the same time when the expression of cyclooxygenase-2 (COX-2) was observed under these conditions. These effects mediated by 15dPGJ2 might contribute to understand the role of COX-2 metabolites in carcinogenesis, leading to an increase of cell viability after mutagenic injury and therefore in the progression of tumors.     

COX-2 dependent regulation of mechanotransduction in human breast cancer cells

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7<SUM-149<MDA-MB-231). Compared with non-invasive MCF-7 and moderately-invasive SUM-149, poorly-differentiated MDA-MB-231 cells showed increased cellular dispersion on collagen matrix that was accompanied by emergent distribution of contractile stresses at the interface between the adherent cell and its substrate, defined herein as the traction field. In metastatic MDA-MB-231 cells, the local tractions were precisely tuned to the surrounding matrix rigidity in a physiologic range with the concomitant expression of mechanosensitive integrin β₁. These discrete responses at the single-cell resolution were correlated with PGE₂ secretion and were ablated by shRNA-mediated knockdown of COX-2. Both COX-2-silenced and COX-2-expressing cells expressed EP2 and EP4 receptors, but not EP1 and EP3. Exogenous addition of PGE₂ increased cell tractions and stiffened the underlying cytoskeletal network. To our knowledge this is the first report linking the expression of COX-2 with mechanotransduction of human breast cancer cells, and the regulation of COX-2-PGE₂-EP signaling with physical properties of the tumor microenvironment. Drug treatments aimed at reducing this mechanical interplay may have therapeutic potential in the treatment of breast cancer.     

Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention.

Interest in chemoprevention in oncology using suppressants of prostaglandin (PG) synthesis has been stimulated by epidemiological observations that the use of aspirin and other non-steroidal inflammatory drugs (NSAIDs) is associated with reduced incidence of some cancers, including cancer of the breast. The main target of NSAID activity is the cyclooxygenase (COX) enzyme. Two isoforms of COX have been identified: COX-1, the constitutive isoform; and COX-2. the inducible form of the enzyme. COX-2 can undergo rapid induction in response to many factors such as bacterial lipopolysaccharides, growth factors, cytokines and phorbol esters. COX-2 is overexpressed in some malignancies including carcinoma of the breast. It has been suggested that such enhanced expression may lead to increased angiogenesis such that the inhibition of COX-2 might have a general anticancer effect via decreased blood vessel formation. In addition, an association between COX-2, its main product PGE2 and aromatase activity in human breast cancer suggests that such inhibitors might have an additional, specific prophylactic mechanism for this tumour. New COX-2 inhibitors are already licensed for use in the treatment of arthritis and are well tolerated. Their potential role in chemoprevention of mammary carcinogenesis in rats has already been investigated. What remains to be seen is if these findings can be extrapolated to human studies.     

Celecoxib induces apoptosis in COX-2 deficient human gastric cancer cells through Akt/GSK3beta/NAG-1 pathway

In this study, we analyzed the mechanisms of the apoptotic effects of celecoxib on COX-2 deficient gastric cancer cell line, MGC-803. We found celecoxib treatment induced caspase-dependent apoptosis in MGC-803 cells. Celecoxib inhibited Ser473 Akt and Ser9 GSK3beta phosphorylation and induced upregulation of nonsteroidal anti-inflammatory drugs-activated gene-1 (NAG-1) expression. The effects of celecoxib on NAG-1 expression were abolished by pretreatment with GSK3beta inhibitor, SB216763. Furthermore, GSK3beta gene silencing by siRNA inhibited the celecoxib-induced NAG-1 expression. Our study demonstrated that Akt/GSK3beta/NAG-1 signal pathway may represent as the major mechanism of the COX-2-independent effects of celecoxib on gastric cancer cells.     

Mullerian-inhibiting substance induces Gro-beta expression in breast cancer cells through a nuclear factor-kappaB-dependent and Smad1-dependent mechanism

Mullerian-inhibiting substance (MIS), a transforming growth factor-beta family member, activates the nuclear factor-kappaB (NF-kappaB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-beta. Inhibiting NF-kappaB activation with a phosphorylation-deficient IkappaBalpha mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-beta promoter-driven reporter expression and Gro-beta mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-beta mRNA, suggesting that regulation of Gro-beta expression by MIS was dependent on activation of NF-kappaB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of kappaB-binding sequences within Gro-beta, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-kappaB complex bound to the kappaB site of the Gro-beta promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-beta promoter in vivo, whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-kappaB signaling in which enhancement of NF-kappaB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the kappaB consensus site within the promoter.