Expression of the proteolysis-inducing factor core peptide mRNA is upregulated in both tumour and adjacent normal tissue in gastro-oesophageal malignancy

Gastro-oesophageal cancer is associated with a high incidence of cachexia. Proteolysis-inducing factor (PIF) has been identified as a possible cachectic factor and studies suggest that PIF is produced exclusively by tumour cells. We investigated PIF core peptide (PIF-CP) mRNA expression in tumour and benign tissue from patients with gastro-oesophageal cancer and in gastro-oesophageal biopsies for healthy volunteers. Tumour tissue and adjacent benign tissue were collected from patients with gastric and oesophageal cancer (n = 46) and from benign tissue only in healthy controls (n = 11). Expression of PIF-CP mRNA was quantified by real-time PCR. Clinical and pathological information along with nutritional status was collected prospectively. In the cancer patients, PIF-CP mRNA was detected in 27 (59%) tumour samples and 31 (67%) adjacent benign tissue samples. Four (36%) gastro-oesophageal biopsies from healthy controls also expressed PIF-CP mRNA. Expression was higher in tumour tissue (P = 0.031) and benign tissue (P = 0.022) from cancer patients compared with healthy controls. In the cancer patients, tumour and adjacent benign tissue PIF-CP mRNA concentrations were correlated with each other (P<0.0001, r = 0.73) but did not correlate with weight loss or prognosis. Although PIF-CP mRNA expression is upregulated in both tumour and adjacent normal tissue in gastro-oesophageal malignancy, expression does not relate to prognosis or cachexia. Post-translational modification of PIF may be a key step in determining the biological role of PIF in the patient with advanced cancer and cachexia. © 2006 Cancer Research.

The role of eicoapentaenoic acid in cancer cahexia

Cachexia is a wasting syndrome often associated with malignancy, characterised by alterations in host metabolism and significant catabolism of host adipose tissue and skeletal muscle. The MAC16 murine adenocarcinoma is profoundly cachexigenic, inducing host weight-loss at relatively small tumour burden without the induction of anorexia. A 4DkDa factor capable of inducing lipolysis in vitro via an activation of adenylate cyclase (AC) has been isolated from the MAC16 tumour, and the urine of cachectic cancer patients, using a series of ion exchange and gel exclusion chromatography procedures. This lipid-mobilising factor (LMF) has been demonstrated to stimulate lipolysis in adipocytes dose-dependently via a signal transduction pathway involving, possibly, β3-adrenoceptors. Oral administration of the n-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) attenuated the progression of cachexia, but not the production of LMF, in MAC16 tumour-bearing mice, and was significantly incorporated into plasma phospholipids, skeletal muscle and adipose tissue. EPA supplemented cancer patients also demonstrated significantly increased plasma EPA concentrations. Decreased plasma membrane AC activity in response to LMF was observed in adipocytes isolated from mice receiving EPA. Incubation in vitro of adipocytes, or plasma membranes, with PUFAs significantly altered membrane fatty acid composition and attenuated the induction of both lipolysis, and AC activity, by LMF. The inhibitory actions of EPA, but not docosahexaenoic acid, are probably the consequence of an interaction with guanine nucleotide binding proteins (G-proteins). Progression of the cachectic state induced an up-regulation of adipocyte membrane expression of stimulatory G-proteins, allied with a concomitant down-regulation of inhibitory G-proteins, thus facilitating the catabolic actions of LMF, implying some tumour-mediated effect. A reversal of such alterations was observed upon oral administration of EPA, suggesting that the primary mechanism of action of this fatty acid is an inhibition of the end organ effects of LMF.

Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

Oxidative catabolism of tetrahydropterins

Mixed labelled folic acid was administerd to rats. Exposure to N2O was used to give an insight into the major route of scission within the monoglutamate pool, results suggest that THF formed during transport from the gut lumen to the plasma is the major route of scission within the gut. Peroxides in corn oil and arising as a result of lipid peroxidation and autoxidation increase catabolism of the monoglutamate pool and decrease incorporation of administered folates into the polyglutamate pool. It is suggested that peroxides may oxidise B12 resulting in inhibition of methionine synthetase, this results in diminished polyglutamation and increased urinary excretion of 5 CH3THF. Fats undergo peroxidation within tissues, the resulting peroxides increase catabolism of the polyglutamate pool. It is suggested that the NBT assay may reflect polyglutamate breakdown. Antioxidants such as vitamin E (and DES) decrease catabolism of the monoglutamate pool. Administration of DES resulted in changes similar to those observed during malignancy, it is suggested that these changes may precede the onset of tumour development. Vitamin E elevates brain DHPR activity. Since lowered DHPR levels and disturbed THB metabolism have been observed in aging and Down's syndrome it is proposed that vitamin E therapy may prove beneficial in situations where oxidative stress is increased. Brain DHPR activity was increased on administration of peroxides suggesting that in situations of oxidative stress (which may result in increased catabolism of THB) the salvage pathway may be stimulated and loss of THB minimised. N2O exposure had no effect on THB metabolism suggesting that the stimulatory role of 5 CH3THF is due to its role as a methyl donor.

Studies of the mechanism of antitumour activity of N-methylformamide

NMF induces the terminal differentiation or acquisition of more benign characteristics in certain malignant cells in vitro and has good antitumour activity against murine tumours in vivo. This study was concerned with a comparison of the mechanism of antitumour activity of NMF in vitro and in vivo against the murine TLX5 lymphoma, which is sensitive to NMF in vivo. TLX5 cells incubated continuously with NMF in vitro showed a concentration and time dependent decrease in cell growth rate, which was associated with an increase in membrane permeability, a decrease in cell size and at the higher NMF concentrations, cell death. Analysis of the cell cycle after incubation with NMF indicated an early G1 phase arrest. TLX5 cells were incubated with NMF and washed free of the drug. Analysis of clonogenicity and tumourigenicity showed that all viable cells retained their proliferative potential and malignancy. Therefore, TLX5 cells exposed to NMF in vitro are not terminally differentiated, but reside in a quiescent substate which was reversed on drug removal. The intracellular GSH levels of TLX5 cells was decreased in a concentration and time dependent fashion by NMF. GSH depletion of TLX5 cells was not however a prerequisite for growth arrest, unlike the reported data for human colon carcinoma cell lines. A single administration of NMF caused a dose dependent regression of the TLX5 lymphoma in tumour bearing mice. Cell death occurred by apoptosis and necrosis. The antitumour activity of NMF was dependent on formyl C-H bond fission, with the parent drug or metabolites reaching all parts of the tumour 4h after dosing. There was a non-dose dependent increase in the S phase population, which was due to an increase in DNA synthesis, 24h after administration of NMF. NMF administration caused a decrease in GSH levels of the TLX5 lymphoma, which did not correlate with the antitumour response. However, the GSH depleting agent, BSO, marginally increased the antitumour activity of NMF.

Bromelain's activity and potential as an anti-cancer agent:Current evidence and perspectives

The medicinal qualities of pineapple are recognized in many traditions in South America, China and Southeast Asia. These qualities are attributed to bromelain, a 95%-mixture of proteases. Medicinal qualities of bromelain include anti-inflammatory, anti-thrombotic, fibrinolytic and anti-cancer functions. Existing evidence derived from clinical observations as well as from mouse- and cell-based models suggests that bromelain acts systemically, affecting multiple cellular and molecular targets. In recent years, studies have shown that bromelain has the capacity to modulate key pathways that support malignancy. It is now possible to suggest that the anti-cancer activity of bromelain consists in the direct impact on cancer cells and their micro-environment, as well as in the modulation of immune, inflammatory and haemostatic systems. This review will summarize existing data relevant to bromelain's anti-cancer activity and will suggest mechanisms which account for bromelain's effect, in the light of research involving non-cancer models. The review will also identify specific new research questions that will need to be addressed in order for a full assessment of bromelain-based anti-cancer therapy.

The Role of Aquaporin 3 (AQP3) in Breast Cancer

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention.

Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy:effects of laser photocoagulation and angiotensin receptor blockade

Background: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. Methods: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. Results: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. Conclusion: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.