Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells.

BACKGROUND: Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved. METHODS: The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference. RESULTS: Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells. CONCLUSION: The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.

BAG-1 predicts patient outcome and tamoxifen responsiveness in ER-positive invasive ductal carcinoma of the breast.

BAG-1 (bcl-2-associated athanogene) enhances oestrogen receptor (ER) function and may influence outcome and response to endocrine therapy in breast cancer. We determined relationships between BAG-1 expression, molecular phenotype, response to tamoxifen therapy and outcome in a cohort of breast cancer patients and its influence on tamoxifen sensitivity in MCF-7 breast cancer cells in vitro. Publically available gene expression data sets were analysed to identify relationships between BAG-1 mRNA expression and patient outcome. BAG-1 protein expression was assessed using immunohistochemistry in 292 patients with invasive ductal carcinoma and correlated with clinicopathological variables, therapeutic response and disease outcome. BAG-1-overexpressing MCF-7 cells were treated with antioestrogens to assess its effects on cell proliferation. Gene expression data demonstrated a consistent association between high BAG-1 mRNA and improved survival. In ER+ cancer (n=189), a high nuclear BAG-1 expression independently predicted improved outcome for local recurrence (P=0.0464), distant metastases (P=0.0435), death from breast cancer (P=0.009, hazards ratio 0.29, 95% CI: 0.114-0.735) and improved outcome in tamoxifen-treated patients (n=107; P=0.0191). BAG-1 overexpression in MCF-7 cells augmented antioestrogen-induced growth arrest. A high BAG-1 expression predicts improved patient outcome in ER+ breast carcinoma. This may reflect both a better definition of the hormone-responsive phenotype and a concurrent increased sensitivity to tamoxifen.

Coracoid impingement syndrome: a treatable cause of anterior shoulder pain.

BACKGROUND: Coracoid Impingement Syndrome is a relatively uncommon but generally treatable cause of anterior shoulder pain that can be easily overlooked. It typically presents with anterior shoulder joint pain in activities involving forward flexion, adduction and internal rotation. AIMS: To assess the outcome of a cohort of patients diagnosed with Coracoid Impingement Syndrome. METHODS: Patients were investigated clinically and radiologically. They received appropriate therapeutic measures and were followed-up in an orthopaedic outpatient setting. RESULTS: Twelve patients were identified over a four-year period. All patients have made good progress. Thus far, none have needed operative intervention for symptom relief. CONCLUSION: Coracoid impingement syndrome is an uncommon cause of anterior shoulder pain but diagnosed patients can expect good symptomatic relief following referral to a dedicated shoulder unit. An increase in clinical awareness of the condition may prevent undue diagnostic delay in such cases.

Butyrate- but not TGFbeta1-induced apoptosis of colorectal adenoma cells is associated with increased expression of the differentiation markers E-cadherin and alkaline phosphatase.

Sodium butyrate and transforming growth factor beta (TGFbeta1) are growth inhibitory to colonic adenoma cell lines. Butyrate induces apoptosis, whereas in some adenoma cell lines, TGFbeta1 can be growth inhibitory without apoptosis. In this report, we show that the adenoma cell line PC/BH/C1 undergoes apoptosis in response to TGFbeta1. Butyrate induced cell death is preceded by the induction of two markers of colonic differentiation--alkaline phosphatase (ALP) activity and E-cadherin protein expression. However, TGFbeta1-induced apoptosis was not accompanied by induction of these differentiation markers. It is possible that the apoptosis induced by TGFbeta1 in the adenoma cell line PC/BH/C1 is due to conflicting signals, as downregulation of c-myc protein in response to TGFbeta1 occurs only slowly in this cell line. Development of resistance to TGFbeta1 in colonic tumours may involve two separate stages--resistance to growth inhibition and resistance to TGFbeta1-induced apoptosis. Our results indicate that sodium butyrate induces apoptosis via differentiation, but TGFbeta1 induces apoptosis by a differentiation-independent mechanism. As for butyrate, the induction of E-cadherin expression is a potentially important chemopreventative action, since increased E-cadherin expression has been correlated with decreased metastatic potential. This is the first report of induction of E-cadherin by a naturally occurring factor in the diet. Butyrate may reduce tumour growth and invasion, not only as a result of the induction of apoptosis, but also through increased expression of E-cadherin.

Sciatic nerve palsy secondary to postoperative haematoma in primary total hip replacement.

Sciatic nerve palsy is a recognised complication of primary total hip replacement. In our unit this complication was rare with an incidence of < 0.2% in the past ten years. We describe six cases of sciatic nerve palsy occurring in 355 consecutive primary total hip replacements (incidence 1.69%). Each of these palsies was caused by post-operative haematoma in the region of the sciatic nerve. Cases, which were recognised early and surgically-evacuated promptly, showed earlier and more complete recovery. Those patients for whom the diagnosis was delayed, and who were therefore managed expectantly, showed little or no recovery. Unexpected pain and significant swelling in the buttock, as well as signs of sciatic nerve irritation, suggest the presence of haematoma in the region of the sciatic nerve. It is, therefore, of prime importance to be vigilant for the features of a sciatic nerve palsy in the early post-operative period as, when recognised and treated early, the injury to the sciatic nerve may be reversed.

Use of Gamma locking nail(®) in tri-planar osteotomy in bilateral severe slipped upper femoral epiphysis: a case study and literature review.

INTRODUCTION: Slipped upper femoral epiphysis (SUFE) is a common condition affecting adolescent boys and girls. It is classified as acute, chronic or acute on chronic. The slip can be mild, moderate or severe. MATERIALS AND METHODS: We present a case of chronic severe SUFE in a 16-year-old male with significant fixed bilateral deformities requiring osteotomy of proximal femur and stabilisation with short locking Gamma nail(®). To our knowledge, this device has not been used in stabilisation of osteotomies in chronic SUFE. CONCLUSION: The purpose of this paper is to describe the results of our fixation method and also to increase the awareness in orthopaedic surgeon about the usefulness of Gamma locking nail(®) in these difficult situations.

Identification of PUMA as an estrogen target gene that mediates the apoptotic response to tamoxifen in human breast cancer cells and predicts patient outcome and tamoxifen responsiveness in breast cancer.

Recognition of the pivotal role of estrogen in the aetiology of breast cancer has led to the development of antiestrogens (AE), such as tamoxifen (TAM) as effective therapies for the treatment and prevention of this disease. However, despite their widespread clinical efficacy, response to AEs is often short-lived, and acquired or innate therapeutic resistance remains a major obstacle in the successful treatment of breast cancer. Thus, delineating the intracellular pathways that mediate the cellular response to estrogen could potentially lead to new, more effective approaches to the treatment of breast cancer, particularly endocrine-resistant disease. Here, we have identified the BCL-2 homology 3 (BH3)-only, pro-apoptotic regulator, PUMA (p53 upregulated modulator of apoptosis) as an estrogen target gene that is acutely downregulated in response to estrogen in breast cancer cell lines, independently of their p53 status. PUMA is transcriptionally upregulated following treatment with TAM, and knock down of PUMA expression in these cells attenuates the apoptotic response to TAM. Furthermore, low PUMA expression in breast carcinomas is significantly associated with breast cancer-specific death (P=0.0014 and P=0.0115, for mRNA and protein, respectively), and worse outcome in TAM-treated patients (mRNA, P=1.49e-05). These findings suggest that the dysregulation of apoptotic signaling pathways such as those executed through PUMA, can significantly impact on both the progression and therapeutic responsiveness of breast cancer. Moreover, they provide a convincing rationale for exploring new therapeutic approaches involving endocrine and non-endocrine therapies that target apoptotic pathways as an effective strategy for tackling endocrine refractory disease.

BAG-1 overexpression attenuates luminal apoptosis in MCF-10A mammary epithelial cells through enhanced RAF-1 activation.

Although the multi-functional, prosurvival protein, Bcl-2-associated anthanogene 1 (BAG-1) is frequently overexpressed in breast cancers, its role in the development or maintenance of the malignant state remains unclear. Here, we have used the established MCF-10A 3-dimensional (3D) model of mammary morphogenesis as a biologically relevant system to determine how BAG-1 expression may influence the development of breast cancer. When cultured in 3D, MCF-10A cells undergo a highly regulated morphogenic program leading to the development of polarized acinar structures containing a central, hollow lumen formed, in part, through the induction of BIM-dependent apoptosis. BAG-1 overexpression resulted in an attenuation of this normal apoptotic program characterized by a significantly increased number of acini with filled lumens-a phenotype commonly observed in ductal carcinoma in situ. BAG-1's effects were associated with an activation of RAF-1-a known binding partner of BAG-1, enhanced signaling through the MAP kinase pathway and a decrease in BIM expression. Reversal of the BAG-1-associated survival phenotype by the mitogen-activated kinase/ERK kinase inhibitor, U0126, implicates the RAF-1-extracellular signal-regulated kinase signaling pathway as a major mediator of BAG-1's effects in this model. As BAG-1 expression is often elevated in preinvasive breast cancers, these findings support a possible role for BAG-1 as an early contributor to the malignant process in the breast.

IGFBP-3 and apoptosis--a license to kill?

Insulin-like growth factor binding protein (IGFBP)-3, the major carrier of insulin-like growth factors (IGFs) in the circulation, was first isolated and characterised over a decade ago. More recently, IGFBP-3 has been assigned a role as a putative death-promoting factor, a function that appears, under certain circumstances, to be independent of its IGF-binding ability. This review examines the current evidence for a pro-apoptotic function for IGFBP-3 and speculates on its physiological significance.

The IGF axis and programmed cell death.

Insulin-like growth factors (IGF) are mitogenic peptides that have been implicated as positive regulators of cellular proliferation. In recent years, several studies have suggested an additional role for the IGF axis in the regulation of apoptosis. Signalling through the IGF receptor has been shown to have a potent survival function and protect cells from a variety of apoptotic stimuli. The actions of IGF are regulated by a family of high-affinity IGF binding proteins (IGFBP), which sequester the IGF from the IGF receptor. However, there is some evidence that one of these binding proteins, IGFBP-3, may have its own pro-apoptotic effects that are independent of its ability to modulate IGF bioavailability. In addition, it has been suggested that the tumour suppressor p53, a crucial mediator of apoptosis in response to cellular stress, may elicit several of its apoptotic effects through manipulation of components of the IGF axis. This review summarizes what is currently known about the role of the IGF system in the regulation of apoptosis, highlighting its implications in the context of tumorigenesis.

Functional activation of Nedd2/ICH-1 (caspase-2) is an early process in apoptosis.

The ICE/CED-3 family of proteases (caspases) play a central role in the execution phase of apoptosis. These proteases are synthesised as precursor molecules that require processing at specific aspartate residues to produce the two subunits that comprise the active enzyme. The activation of some of these proteases has been shown to occur during apoptosis. Here we show that Nedd2/ICH-1 (caspase-2) is activated during apoptosis induced by a variety of apoptotic stimuli. This activation occurs very early upon treatment of cells with apoptotic agents and appears to precede the activation of CPP32 (caspase-3). The activation of Nedd2 was not seen in cells that are resistant to apoptosis. These observations suggest that Nedd2 is an early effector in the pathway leading to cell death. Our observations also lend weight to the hypothesis that a group of caspases containing long prodomains are the first to be activated in response to apoptotic signals and that they lie upstream of a second class of caspases such as CPP32 containing short or no prodomains.

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells.

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

Dimerization and autoprocessing of the Nedd2 (caspase-2) precursor requires both the prodomain and the carboxyl-terminal regions.

Nedd2 (caspase-2) is a cysteine protease of the caspase family that has been demonstrated to play a role in the apoptotic pathway. The 51-kDa precursor of Nedd2 undergoes cleavage into two subunits following various apoptotic stimuli. In this study, we have investigated the dimerization of the Nedd2 precursor (pro-Nedd2) in Saccharomyces cerevisiae and its self-processing activity in vivo. We demonstrate that the expression of pro-Nedd2 in yeast cells results in processing of the precursor. A catalytically inactive pro-Nedd2 mutant dimerized in yeast, and the dimerization required both the prodomain and the carboxyl-terminal residues. Aspartate mutants that block the removal of the p14/p12 subunits, but not the wild-type Nedd2, were shown to dimerize in yeast cells, suggesting that dimerization occurs prior to processing. In vitro processing of pro-Nedd2 by recombinant active Nedd2 defined the aspartate residues that are crucial for processing to occur. Both the in vivo and in vitro processing of pro-Nedd2 directly correlated with its ability to induce cell death in transient overexpression experiments.

Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular-weight form is associated with apoptosis in vitro.

APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival.

Growth inhibition by insulin-like growth factor-binding protein-3 in T47D breast cancer cells requires transforming growth factor-beta (TGF-beta ) and the type II TGF-beta receptor.

This study explores the relationship between anti-proliferative signaling by transforming growth factor-beta (TGF-beta) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-beta1. To investigate the mechanism, we used T47D cells that lack type II TGF-beta receptor (TGF-betaRII) and are insensitive to TGF-beta1. After introducing the TGF-betaRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-beta1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-beta1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-betaRII-expressing cells when exogenous TGF-beta1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-beta1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-betaRII and exogenous TGF-beta1. To investigate this synergism, the phosphorylation of TGF-beta signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-beta1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-beta signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.

Transcriptional upregulation of the insulin-like growth factor binding protein IGFBP-3 by sodium butyrate increases IGF-independent apoptosis in human colonic adenoma-derived epithelial cells.

Sodium butyrate (NaBt) and the pro-apoptotic IGFBP-3 protein, expressed at the top of the normal colonic crypt, have both been implicated in the regulation of apoptosis in colonic epithelial cells. Recent studies in human breast and hepatic cell lines have shown that NaBt can transcriptionally upregulate IGFBP-3 expression. However, the role of butyrate in the regulation of IGFBP-3 expression in the colon is less clear, with reports of both up- and downregulation of the IGFBP-3 protein in colorectal cancer cell lines. In this study we have shown that the level of IGFBP-3 protein expression in colonic epithelial cells correlates with the p53 status of the cells; wildtype p53 cells secrete higher levels of IGFBP3 protein than mutant p53 cell lines. Data presented shows that, when treated with a dose of NaBt that induced significant apoptosis (4 mM for 48 h), there was an upregulation of IGFBP-3 protein in both wildtype and mutant p53 expressing cell lines. The NaBt-induced increase in secreted IGFBP-3 protein was associated with transcriptional upregulation of the IGFBP-3 gene. Using a transfected derivative of the S/RG/C2 adenoma-derived cell line, which stably expressed exogenous IGFBP-3 protein at levels equivalent to that secreted by the 4 mM NaBt-treated parental line (1-3 ng/10(6) cells), we have shown a >2-fold increase in the sensitivity of the cells to NaBt-induced apoptosis when compared with the vector control and parental cell lines. Furthermore, inhibition of the secreted IGFBP-3 protein, by addition of neutralizing antibodies, resulted in a significant decrease in NaBt-induced apoptosis. These data suggest that IGFBP-3 may act as a positive regulator of NaBt-induced apoptosis in colonic epithelial cells, and represents a potentially important mechanism whereby the sensitivity of colonic epithelial cells to NaBt-induced apoptosis can be increased.