Biomedical subjects

B K Tsang

Publications and source records attributed to B K Tsang.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of p53 and suppression of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian cancer cells.

Dysregulated apoptosis plays a critical role in the development of a number of aberrant cellular processes, including tumorigenesis and chemoresistance. However, the mechanisms that govern the normal apoptotic program are not completely understood. Soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP) promote mammalian cell viability via an unknown mechanism and p53 status is a key determinant of cell fate in human ovarian cancer cells. Whether an interaction exists between these two determinants of cell fate is unknown. We hypothesized that basal sGC activity reduces p53 content and attenuates p53-dependent apoptosis in human ovarian cancer cells. Suppression of sGC activity with the specific inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) lowered cGMP content, and increased p53 protein content and induced apoptosis in three ovarian cancer cell lines, effects which were attenuated by the cGMP analog 8-Br-cGMP and by Atrial Natriuretic Factor, an activator of particulate guanylyl cyclase, which circumvent the inhibition of sGC. ODQ prolonged p53 half-life, induced phosphorylation of p53 on Ser15, and upregulated the p53-dependent gene products p21, murine double minute-2, and the proapoptotic, p53-responsive gene product Bax. ODQ activated caspase-3, and ODQ-induced apoptosis was inhibited by overexpression of X-linked inhibitor of apoptosis Protein. Pretreatment with the specific p53 inhibitor pifithrin or downregulation of p53 using a specific small inhibitory RNA significantly attenuated ODQ-induced apoptosis. Moreover, ODQ-induced upregulation of p21 and Bax and ODQ-induced apoptosis were significantly reduced in a p53 mutant cell line relative to the wild-type parental cell line. Thus, the current study establishes that basal sGC/cGMP activity regulates p53 protein stability, content, and function, possibly by altering p53 phosphorylation and stabilization, and promotes cell survival in part through regulation of caspase-3 and p53.

Adenoviridae↗

PubMed2005

Comparisons of brain, uterus, and liver mRNA expression for cytochrome p450s, DNA methyltransferase-1, and catechol-o-methyltransferase in prepubertal female Sprague-Dawley rats exposed to a mixture of aryl hydrocarbon receptor agonists.

Non-ortho polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) are ubiquitous environmental contaminants that exert their toxicity mostly through activation of the aryl-hydrocarbon receptor (AhR), and are referred to as AhR agonists. The objective was to study, by real time reverse-transcriptase-polymerase chain reaction (RT-PCR), the effects of postnatal exposure to a reconstituted mixture of AhR agonists present in breast milk (3 non-ortho PCBs, 6 PCDDs, and 7 PCDFs, referred to here-in-after as AhRM) on mRNA expression of estrogen receptor (ERalpha), enzymes involved with the metabolism of estrogens [catechol-o-methyltransferase (Comt), cytochrome P450 (Cyp)1A1, 1B1 and 2B1], and DNA methyltransferase-1 (Dnmt1), in brain areas, liver and uterus of immature female rats. Neonates were exposed by gavage during postnatal day (PND) 1-20 with dosages equivalent to 1, 10, 100, and 1000 times the estimated average human exposure level, and were sacrificed at PND 21. None of the end points were affected in uterine cross-sections, or in samples of uterine tissue layers collected by laser capture microdissection. At 1000x, the AhRM reduced Dnmt1 mRNA abundance to 28% and 32% of control in the liver and hypothalamus, respectively. In the brain, Cyp1A1 was increased (409%) but ERalpha was reduced (66%). Similarly, mRNA abundance for Comt isoforms was reduced in the liver (45%) and brain areas (55-70%). AhRM at 100x, the lowest effective dose, exerted a 220% increase in brain cortex Comt [membrane bound (Mb)], a 219% increase in hepatic Cyp1B1, and a 63% decrease in hepatic Comt (soluble (S)+Mb). These results support the possibility that early exposure to environmental contaminants could lead to effects mediated by changes in DNA methylation and/or estrogen metabolism and signaling.

Animals↗

PubMed2004

Role of Akt/protein kinase B in the activity of transcriptional coactivator p300.

Akt/protein kinase B is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway and plays a critical role in promotion of cell survival. The function of transcriptional coactivator p300 is required by many transcription factors to either activate or repress gene expression. Here, we show that induction of PI3K enhances the metabolic stability of endogenous p300 protein. On the other hand, repression of PI3K by LY294002 induces p300 degradation through the 26S proteasome pathway and impedes the transcriptional activity of the coactivator. In addition, Akt interacts with the coactivator and the activity of Akt is required to maintain the steady-state level of p300. Our study provides a new insight into the molecular mechanisms by which the critical concentration of p300 protein is regulated and suggests a role for Akt in control of various cellular activities through the transcriptional coactivator p300.

Acetyltransferases↗

PubMed2004

EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways.

Epidermal growth factor (EGF) is present in the maternal-fetal environment and has an important role in placental development. Matrix metalloproteinase-9 (MMP-9) expression/activation is a pre-requisite in extravillous trophoblast invasion. Whereas EGF up-regulates MMP-9 activity in a variety of cell types, there is no direct evidence for the stimulation of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion by EGF in extravillous trophoblasts. In addition, the signalling pathways involved in this regulation are not clear. In the present study, we have examined the possible involvement of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in the regulation of the MMP-9/TIMP-1 system by EGF in vitro. We used a well-established invasive extravillous trophoblast cell line (HTR8/Svneo) and measured gene and protein expression by semi-quantitative RT-PCR and western analysis respectively. MMP activity was determined by zymography. We showed for the first time that EGF activated both PI3K/Akt and MAPK/extracellular-signal regulated kinase (ERK) signalling in HTR8/SVneo, and increased both MMP-9 and TIMP-1 mRNAs and protein concentrations. Interfering with either signalling pathway via PI3K inhibitor LY294002 or MEK inhibitor U0126 in EGF-stimulated HTR8/SVneo cells blocked the induction of MMP-9 and TIMP-1. LY294002 inhibited Akt phosphorylation, but had no effect on ERK phosphorylation; U0126 suppressed ERK phosphorylation without interfering with the phosphorylation of Akt. In addition, expression of constitutively active Akt (Myr-Akt1, Myr-Akt2, Myr-Akt3) was not sufficient to induce proMMP-9 and TIMP-1 secretion. Our results suggest that the activation of both PI3K and MAPK pathways in extravillous trophoblasts is necessary for the up-regulation of MMP-9 and TIMP-1 expression by EGF.

Blotting, Western↗

PubMed2003

Effects of postnatal exposure to mixtures of non-ortho-PCBs, PCDDs, and PCDFs in prepubertal female rats.

There are concerns that postnatal exposure to organochlorines present in breast milk could lead to adverse health effects. We reconstituted four mixtures of aryl-hydrocarbon receptor (AhR) agonists (3 non-ortho polychlorinated biphenyls [PCBs], 6 polychlorinated dibenzodioxins [PCDDs], 7 polychlorinated dibenzofurans [PCDFs], or all 16 chemicals together [referred to as AhRM]) based on their concentrations in breast milk, and examined their effects following exposure by gavage from day 1 until day 20 of age. Female neonates received dosages of AhRM equivalent to 1, 10, 100, or 1000 times the amount consumed by an infant over the first 24 days of life. Other groups received the PCBs, the PCDDs, or the PCDFs at the 1000x level. All rats were sacrificed at 21 days of age. Changes in ethoxyresorufin-o-deethylase hepatic activity, thymus and body weights, and serum thyroxin were linked to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalents (TEQ) of the four mixtures (1000x-AhRM > PCDDs > PCBs > PCDFs). To test for AhRM antiestrogenicity, two additional groups received 1.5 microg/kg of 17alpha-ethynyl estradiol (EE) with or without the 1000x-AhRM. The AhRM had no effect on uterine weight or EE-stimulated uterine growth. The actions of the combined EE and AhRM treatments suggest additive effects in decreasing pentoxyresorufin-o-deethylase activity and spleen weight, but nonadditive/antagonistic effects on adrenal weight and serum thyroxin. In conclusion, (1) 10x-AhRM had no detectable effects, (2) TEQ values relate to observed toxicities, even when testing complex mixtures of AhR agonists, and (3) indications of tissue-specific additive and nonadditive/antagonistic effects, but no synergism, were observed when doses of AhRM were increased, or combined with EE.

Animals↗

PubMed2003

Capillary angiogenesis and degeneration in bovine ovarian antral follicles.

Angiogenesis and capillary degeneration are both evident during ovarian follicle growth. However, the characteristics and distribution of thecal capillary proliferative and degenerative structures have not been fully defined. Indeed, the role of thecal microvasculature changes in follicular atresia is still a matter of debate. The present study examined the distribution of thecal capillary changes occurring during follicular growth and related the changes to capillary morphology (by scanning electron microscopy, SEM, on bovine ovarian corrosion casts) with the incidence of capillary apoptosis (TdT-mediated dUTP nick end-labelling, TUNEL) and follicular status (as confirmed by follicular fluid steroid concentrations). SEM demonstrated well-perfused vascular plexuses of small to large antral follicles with structural and functional changes to capillaries. Angiogenesis was evident mainly in the apical part of the inner capillary layer of medium follicles and the middle or basal part of the inner capillary layer of dominant follicles that exhibited high oestradiol:progesterone ratios. Degenerative capillaries were observed mainly in the outer vascular layers of small follicles, and in the inner and outer vascular layers of medium antral follicles. Although apoptotic structures were present only in the outer capillaries of the theca interna of morphologically healthy antral follicles, atretic follicles showed apoptotic structures in both the outer and inner thecal capillary layers. These results show that angiogenesis increases during bovine follicular growth and occurs unevenly in different inner theca regions of the follicles. The differential angiogenic and degenerative response of theca interna capillaries may reflect differences in the microenvironment of the follicles, which in turn determine the fate of the follicles (continued growth versus atresia).

Animals↗

PubMed2001

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells.

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.

Antineoplastic Agents↗

PubMed2001

Anti-apoptotic proteins, apoptotic and proliferative parameters and their prognostic significance in cervical carcinoma.

The inhibitor of apoptosis proteins (IAP) suppress apoptosis induced by a variety of stimuli. The aims of this study were to: (a) compare the expression of X-linked IAP (Xiap) and Human IAP-2 (Hiap-2) in cervical carcinoma cells and normal cervix, (b) determine the correlation between IAP expression and tumour apoptosis or proliferation, and (c) assess their prognostic significance in cervical carcinomas. Paraffin-embedded tissue sections were retrieved from 77 patients with cervical squamous carcinomas prior to treatments and 47 normal subjects. Tumour apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuracil triphosphate (dUTP) nick-end labelling (TUNEL) and apoptotic index (AI), and the proliferative rate was measured by Ki-67 and mitotic (MI) indices. Immunoreactive Xiap and Hiap-2 were found in both cervical cancer cells and normal tissues. IAP expressions in cancers did not correlate with apoptotic and proliferative parameters, disease stage and patient survival. The lower AI and Ki-67 index were associated with a better survival. In conclusion, the basal expression levels of IAPs have no prognostic significance, but AI and Ki-67 expression are potential prognostic indicators in cervical carcinoma.

Adult↗

PubMed2001

X-linked inhibitor of apoptosis protein expression and the regulation of apoptosis during human placental development.

In this study, we have examined the expression and potential role of X-linked inhibitor of apoptosis protein (XIAP), Fas, and Fas ligand (FasL) in the regulation of apoptosis throughout placental development. Protein expression was determined by Western blot analysis and immunohistochemistry, whereas apoptotic cell death was assessed by DNA fragmentation analysis and TUNEL. The XIAP was present in trophoblast throughout placental development, but its content significantly decreased during late pregnancy, when apoptosis was maximal. The FasL content was low during early placental development but increased coincidentally to the decrease in XIAP during the third trimester. Our data also suggest that placental apoptosis is the culmination of the relative expression of these cell-death and -survival proteins, a phenomenon that is cell type-specific and dependent on cytodifferentiation and the stage of placental development. Moreover, the induction of syncytiotrophoblast apoptosis may involve the concomitant up-regulation of FasL for Fas activation and the removal of downstream inhibition of the apoptotic cascade by XIAP.

Apoptosis↗

PubMed2001

Follicular stage-dependent tumor necrosis factor alpha-induced hen granulosa cell integrin production and survival in the presence of transforming growth factor alpha in vitro.

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNF alpha) and transforming growth factor alpha (TGF alpha) have been suggested. TGF alpha stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNF alpha and its possible interaction with TGF alpha in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNF alpha and TGF alpha interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGF alpha (0.1 or 10 ng/ml), but not TNF alpha (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin beta(3) subunit was also stimulated by TGF alpha in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNF alpha in the presence of maximal stimulatory concentrations of TGF alpha potentiated this response. TGF alpha increased both F5,6 and F1 granulosa cell [(3)H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNF alpha had no effect on [(3)H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNF alpha and TGF alpha than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGF alpha-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNF alpha alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF alpha but not of TNF alpha. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNF alpha and TGF alpha was necrosis and not apoptosis. The current study demonstrates that TNF alpha and TGF alpha interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF alpha; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.

Acridine Orange↗

PubMed2001

Influence of maternal smoking on trophoblast apoptosis throughout development: possible involvement of Xiap regulation.

Maternal smoking is associated with severe perinatal complications and significant placental pathologies with underlying ultrastructural changes. In this study, we examined the influence of maternal smoking on trophoblast apoptosis throughout development and correlated those findings with changes in expression of X-linked inhibitor of apoptosis protein (Xiap) as well as Fas and Fas ligand (FasL). Trophoblast apoptosis was determined by DNA fragmentation and TUNEL. Protein expression was assessed by Western blotting and immunohistochemistry. Maternal smoking was associated with increased trophoblast apoptosis in the first trimester but decreased trophoblast apoptosis near term. Placental Xiap levels decreased significantly throughout development in nonsmokers (P < 0.05) but remained elevated in smokers. Fas and FasL levels did not vary significantly throughout development nor between groups. However, procaspase-3 levels were significantly increased in smokers at term. Our results suggest that maternal smoking has different effects at different stages of trophoblast differentiation and that this is regulated in part through modulations in placental Xiap expression.

Apoptosis↗

PubMed2001

Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins.

The inhibitor of apoptosis proteins (IAPs) constitutes a family of highly conserved apoptosis suppressor proteins that were originally identified in baculoviruses. Although IAP homologs have recently been demonstrated to suppress apoptosis in mammalian cells, their expression and role in human ovarian epithelial cancer and chemotherapy resistance are unknown. In the present study we used cisplatin-sensitive and -resistant human ovarian surface epithelial (hOSE) cancer cell lines and adenoviral antisense and sense complementary DNA expression to examine the role of IAP in the regulation of apoptosis in human ovarian cancer cells and chemoresistance. Antisense down-regulation of X-linked inhibitor of apoptosis protein (Xiap), but not human inhibitor of apoptosis protein-2 (Hiap-2), induced apoptosis in cisplatin-sensitive and, to a lesser extent, in -resistant cells. Cisplatin consistently decreased Xiap content and induced apoptosis in the cisplatin-sensitive, but not cisplatin-resistant, cells. Hiap-2 expression was either unaffected or inhibited to a lesser extent. The inhibition of IAP protein expression and induction of apoptosis by cisplatin was time and concentration dependent. Infection of cisplatin-sensitive cells with adenoviral sense Xiap complementary DNA resulted in overexpression of Xiap and markedly attenuated the ability of cisplatin to induce apoptosis. Immunohistochemical localization of the IAPs in hOSE tumors demonstrated the presence of Xiap and Hiap-2, with their levels being highest in proliferative, but not apoptotic, epithelial cells. These studies indicate that Xiap is an important element in the control of ovarian tumor growth and may be a point of regulation for cisplatin in the induction of apoptosis. These results suggest that the ability of cisplatin to down-regulate Xiap content may be an important determinant of chemosensitivity in hOSE cancer.

Aged↗

PubMed2001

Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor alpha-induced apoptosis.

Although X-linked inhibitor of apoptosis protein (Xiap) is an important intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-alpha (TNFalpha) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFalpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 microM), and/or adenoviral Xiap sense or antisense complementary DNA. TNFalpha alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTPbiotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFalpha alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expression. Down-regulation of Xiap expression by antisense complementary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-kappaB activation by N-acetyl-cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFalpha. The latter phenomenon was readily attenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFalpha death signaling pathway in granulosa cells. Its expression is regulated by the TNFalpha via a nuclear factor-kappaB-mediated mechanism.

Acetylcysteine↗

PubMed2001

X-linked inhibitor of apoptosis protein activates the phosphatidylinositol 3-kinase/Akt pathway in rat granulosa cells during follicular development.

X-linked inhibitor of apoptosis protein (XIAP) in granulosa cells is regulated by gonadotropins during follicular development, although the current understanding of the mechanisms by which XIAP suppressed granulosa cell apoptosis is incomplete. In the present study, we investigated the possible involvement of the phosphatidylinositol 3-kinase (PI 3-K) survival pathway in the regulation of granulosa cell fate. Using a fully characterized in vivo model to study the induction of follicular development and atresia in immature rats, we have demonstrated that gonadotropin treatment increased granulosa cell XIAP and phospho-Akt protein contents and suppressed apoptosis. In addition, gonadotropin withdrawal [equine CG (eCG)-primed rats treated with an anti-eCG antibody] induced granulosa cell apoptosis and significantly decreased ovarian weight. The increased apoptosis was accompanied by marked decreases in XIAP expression and phosphorylation of Akt protein. Infection of granulosa cells from eCG-primed rats with adenoviral sense XIAP [lacZ as a control; multiplicity of infection, 1-5] resulted in XIAP overexpression and increased phospho-Akt content, whereas XIAP antisense expression (multiplicity of infection, 10-40) decreased granulosa cell phospho-Akt level and induced apoptosis. Addition of the specific PI 3-K inhibitor LY294002 to the granulosa cell cultures decreased Akt phosphorylation and induced apoptosis in a dose-dependent manner. Taken together, these results demonstrate for the first time the importance and regulation of the PI 3-K survival pathway by XIAP in the control granulosa cell apoptosis.

Animals↗

PubMed2001

Role of prostaglandins in the suppression of apoptosis in hen granulosa cells by transforming growth factor alpha.

Although transforming growth factor alpha (TGF-alpha) is known to be an important survival factor for granulosa cells, the cellular and molecular mechanisms involved are uncertain. The purpose of the present study was to investigate the possible involvement of prostaglandins in the anti-apoptotic action of TGF-alpha. Hen granulosa cells from healthy prehierarchical follicles (2-6 mm) cultured in serum-free medium underwent spontaneous apoptosis as demonstrated by DNA fragmentation and nuclear chromatin condensation. TGF-alpha (20 ng ml(-1)) stimulated maximum synthesis of prostaglandins (PGE and PGF) in granulosa cells and completely inhibited serum deprivation-induced apoptosis. The addition of an inhibitor of cyclooxygenase (COX; N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) or ibuprofen) or phospholipase A(2) (PLA(2); aristolochic acid, 2-p-amylcinnamoyl amino-4-chlorobenzoic acid (ONO-RS-82) or arachidonyl triflouro methyl ketone (TFMK)), to the culture medium markedly suppressed the TGF-alpha-induced prostaglandin synthesis and significantly increased granulosa cell apoptosis. The apoptotic effect of NS398 and aristolochic acid was completely inhibited by exogenous prostaglandins (PGF(2 alpha), PGE(1), PGE(2)) and arachidonic acid, respectively. However, exogenous prostaglandins failed to inhibit the PLA(2) inhibitor-induced apoptotic DNA fragmentation, implying that in addition to prostaglandins, arachidonic acid or leukotrienes may be important in transducing the anti-apoptotic action of TGF-alpha. In the absence of exogenous TGF-alpha, prostaglandins had no significant influence on granulosa cell apoptosis induced by serum withdrawal. These findings indicate that prostaglandin synthesis is a necessary, but not sufficient, event in the suppression of granulosa cell apoptosis by TGF-alpha. Whether arachidonic acid or leukotrienes are important in the anti-apoptotic action of TGF-alpha in hen granulosa cells remains to be determined.

Alprostadil↗

PubMed2000

Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells.

Cisplatin-centered chemotherapy is a key treatment for ovarian cancer, but resistance to chemotherapeutic agents remains a major cause of treatment failure. Multiple factors are known to contribute to the development of this chemoresistance. Although it has been demonstrated that X-linked inhibitor of apoptosis protein (Xiap) prevents apoptosis by inhibiting effector caspases, if and how it is important in chemoresistance in ovarian cancer has not been studied. The effects of Xiap down-regulation and/or restoration of wild type p53 by recombinant adenovirus infection were examined on four ovarian epithelial cancer cell lines [C13*, A2780-s (wild type p53), A2780-cp (mutant p53), and SKOV3 (null p53)]. Apoptosis and protein expression (e.g., Xiap, caspase-3, p53, MDM2, and p21waf1) were assessed by Hoechst 33258 stain and Western blot, respectively. We demonstrated that Xiap down-regulation following adenoviral antisense expression induces apoptosis in the wild-type p53 cells, but not in the mutated or null cells. Xiap down-regulation resulted in caspase-3 activation, caspase-mediated MDM2 processing, and p53 accumulation. Restoration of wild type p53 in the p53-mutated or -null cells significantly enhanced the proapoptotic effect of Xiap antisense expression. Down-regulation of Xiap induced apoptosis in chemoresistant ovarian cancer cells, a process dependent on p53 status.

Adenocarcinoma↗

PubMed1999

Sustained suppression of Fas ligand expression in cisplatin-resistant human ovarian surface epithelial cancer cells.

Although cisplatin derivatives are first line chemotherapeutic agents for the treatment of ovarian epithelial cancer, chemoresistance is a major therapeutic problem. Although the cytotoxic effect of these agents are believed to be mediated through the induction of apoptosis, the role of the Fas/FasL system in chemoresistance in human ovarian epithelial cancer is not fully understood. In the present study, we have used cultures of established cell lines of cisplatin-sensitive human ovarian epithelial tumours (OV2008 and A2780-s) and their resistant variants (C13* adn A2780-cp, respectively) to asses the role of Fas/FasL system in the chemo-responsiveness of ovarian cancer cell to cisplatin. Cisplatin was effective in inducing the expression of cell-associated Fas and FasL, soluble FasL and apoptosis in concentration and time-dependent fashion in both cisplatin-sensitive cell lines (OV2008 and A2780-s). In contrast, while cisplatin was effective in increasing cell-associated Fas protein content in C13*, it failed to up-regulate FasL (cell-associated and soluble forms) and induce apoptosis, irrespective of concentration and duration of cisplatin treatment. Concentrated spent media for OV208 cultures after cisplatin treatment were effective in inducing apoptosis in C13* cells which was partly inhibited by the antagonistic Fas monoclonal antibody (mAb) suggesting that the soluble FasL present in the spent media was biologically active. In the resistant A2780-cp cells, neither Fas nor FasL upregulation were evident in the presence of the chemotherapeutic agent and apoptosis remained low compared to its sensitive counterpart. Activation of the Fas signalling pathway, by addition to the cultures an agonistic Fas mAb, was equally effective in inducing apoptosis in the cisplatin-sensitive (OV2008) and -resistant variant C13*, although these responses were of lower magnitude compared to that observed with cisplatin in the chemosensitive cells. A significant interaction between cisplatin and agonistic Fas mAb was observed in the apoptotic response in OV2008 and C13* when cultured in the presence of both agents. Immunohistochemistry of human ovarian epithelial carcinomas reveals the presence of Fas in low abundance in proliferatively active cells but in high levels in quiescent ones. Although the expression pattern of FasL in the tumour was similar to tha tof Fas, the protein content was considerably lower. Taken together, these data suggest that the dysregulation of the Fas/FasL system may be an important determinant in cisplatin-resistance in ovarian epithelial cancer cells. Our results are also supportive of the notion that combined immuno- and chemo-therapy (i.e., agonistic Fas mAb plus cisplatin) may provide added benefits in the treatment of both chemo-sensitive and -resistant ovarian tumours.

Journal Article↗

PubMed1999

Acyl chain length-specific ceramide-induced changes in intracellular Ca2+ concentration and progesterone production are not regulated by tumor necrosis factor alpha in hen granulosa cells.

Although tumor necrosis factor alpha (TNF-alpha) has long been known to be a potent inhibitor of gonadotropin-induced cytodifferentiation in the ovaries of a variety of mammalian species, its early signal transduction events are poorly understood. We previously demonstrated that TNF-alpha induces a small, delayed follicular stage-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in hen granulosa cells and promotes carbachol (Cch)-induced mobilization of Ca2+ from intracellular stores in cells otherwise unresponsive to the cytokine. The focus of the current study was to examine the role of ceramide in TNF-alpha-induced Ca2+ regulation. Treatment with exogenous sphingomyelinase (SMase; 50 mU/ml) failed to influence basal [Ca2+]i but increased the magnitude of Cch-induced Ca2+ transients. While C8-ceramide (0.03-30 microM), but not C2-ceramide (0.03-30 microM), mimicked this effect of SMase, challenge with sphingosine (3 microM) resulted in a slow and delayed increase in basal [Ca2+]i. In order to determine whether SMase is activated by TNF-alpha action, changes in sphingomyelin and ceramide concentrations in F1 and F5,6 granulosa cells were determined. SMase activation was not observed after 1-, 5-, 15-, and 60-min incubations with TNF-alpha (1-50 ng/ml) in either F1 or F5,6 cells. Exogenous SMase and C2-ceramide both inhibited LH-induced progesterone production in F1 and F5,6 cells; however, incubation with C8-ceramide resulted in increases in both basal and LH-induced progesterone. In contrast, incubation with TNF-alpha had no effect on either basal or LH-induced steroidogenesis. In conclusion, our findings indicate that although ceramide regulates [Ca2+]i and progesterone secretion, the sphingolipid does not appear to play a role in the action of TNF-alpha in avian granulosa cells. Furthermore, ceramide-mediated responses are highly dependent on acyl chain length, potentially reflecting differences in the abilities of these ceramides to access, bind to, and/or activate ceramide-dependent signal transduction mechanisms. Nonetheless, since TNF-alpha did not increase the production of ceramide, the physiological regulator(s) of these responses remain unknown.

Animals↗