Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 μg/ml for 0-60 min at 37°C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 μg/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.

Adiponectin (15-36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells:role of AMPK and MAPK kinase pathways

Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production.

Phospholipid chlorohydrins cause ATP depletion and toxicity in human myeloid cells

Chlorohydrins of stearoyl-oleoyl phosphatidylcholine (SOPC), stearoyl-linoleoyl phosphatidylcholine, and stearoyl-arachidonyl phosphatidylcholine were incubated with cultured myeloid cells (111,60) for 24 h, and the cellular ATP level was measured using a bioluminescent assay. The chlorohydrins caused significant depletion of cellular ATP in the range 10100 muM. The ATP depletion by the phospholipid chlorohydrins was slightly less than that of 4-hydroxy-2-nonenal, but greater than that of hexanal, trans-2-nonenal, and autoxidised palmitoyl-arachidonoyl phosphatidylcholine. SOPC chlorohydrin was also found to cause loss of viability in U937 cells, and thus phospholipid chlorohydrins could contribute to the formation of a necrotic core in advanced atherosclerotic lesions.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

Discrimination of healthy and cancer cells of the bladder by metabolic state, based on autofluorescence

Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD ("redox ratio") can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.

BRACHYURY confers cancer stem cell characteristics on colorectal cancer cells

Cancer stem cells (CSCs) are initiating cells in colorectal cancer (CRC). Colorectal tumours undergo epithelial to mesenchymal transition (EMT)-like processes at the invasive front, enabling invasion and metastasis, and recent studies have linked this process to the acquisition of stem cell-like properties. It is of fundamental importance to understand the molecular events leading to the establishment of cancer initiating cells and how these mechanisms relate to cellular transitions during tumourigenesis. We use an in vitro system to recapitulate changes in CRC cells at the invasive front (mesenchymal-like cells) and central mass (epithelial-like cells) of tumours. We show that the mesoderm inducer BRACHYURY is expressed in a subpopulation of CRC cells that resemble invasive front mesenchymal-like cells, where it acts to impose characteristics of CSCs in a fully reversible manner, suggesting reversible formation and modulation of such cells. BRACHYURY, itself regulated by the oncogene β-catenin, influences NANOG and other 'stemness' markers including a panel of markers defining CRC-CSC whose presence has been linked to poor patient prognosis. Similar regulation of NANOG through BRACHYURY was observed in other cells lines, suggesting this might be a pathway common to cancer cells undergoing mesenchymal transition. We suggest that BRACHYURY may regulate NANOG in mesenchymal-like CRC cells to impose a 'plastic-state', allowing competence of cells to respond to signals prompting invasion or metastasis. Copyright © 2011 UICC.

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia. © 2003 Wiley-Liss, Inc.

Mechanical stimulation alters pleiotrophin and aggrecan expression by human intervertebral disc cells and influences their capacity to stimulate endothelial migration

Study Design. The influence of mechanical load on pleiotrophin (PTM) and aggrecan expression by intervertebral disc (IVD) cells, and the effects of disc cell conditioned medium on endothelial cell migration was investigated. Objective. To examine possible interactions of mechanical loads and known pro- and antiangiogenic factors, which may regulate disc angiogenesis during degeneration. Summary of Background Data. Pleiotrophin expression can be influenced by mechanical stimulation and has been associated with disc vascularization. Disc aggrecan inhibits endothelial cell migration, suggesting an antiangiogenic role. A possible interplay between these factors is unknown. Methods. The influence of the respective predominant load (cyclic strain for anulus fibrosus and hydrostatic pressure for nucleus pulposus cells) on PTN and aggrecan expression by IVD cells was determined by real-time RT-PCR and Western blotting (PTN only). The effects of IVD cell conditioned medium on endothelial cell migration were analyzed in a bioassay using human microvascular endothelial (HMEC-1) cells. Results. Application of both mechanical loads resulted in significant alterations of gene expression of PTN (+67%, P = 0.004 in anulus cells; +29%, P = 0.03 in nucleus cells) and aggrecan (+42%, P = 0.03 in anulus cells, -25%, P = 0.03 in nucleus cells). These effects depended on the cell type, the applied load, and timescale. Conditioned media of nucleus pulposus cells enhanced HMEC-1 migration, but this effect was diminished after 2.5 MPa hydrostatic pressure, when aggrecan expression was diminished, but not 0.25 MPa, when expression levels were unchanged. Conclusion. Mechanical loading influences PTN expression by human IVD cells. Conditioned media from nucleus pulposus cell cultures stimulated HMEC-1 endothelial cell migration. This study demonstrates that the influence of mechanical loads on vascularization of the human IVD is likely to be complex and does not correlate simply with altered expression of known pro- and antiangiogenic factors.

Antiproliferative effects of carbon monoxide on pancreatic cancer

Background - Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. Methods - In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500 ppm/24 h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35 mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500 ppm 1 h/day; n = 6 in each group). Results - Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p < 0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30–50%, p < 0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p < 0.01), and doubled the survival rates (p < 0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p = 0.006). Conclusion - These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer.

Human intervertebral disc cells promote nerve growth over substrata of human intervertebral disc aggrecan

Study Design. Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. Objective. To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. Summary of Background Data. Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. Methods. Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. Results. Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. Conclusions. Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

Antiproliferative effects of carbon monoxide on pancreatic cancer

Background: Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. Methods: In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500. ppm/24. h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35. mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500. ppm 1. h/day; n=. 6 in each group). Results: Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p<0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30-50%, p<. 0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p<0.01), and doubled the survival rates (p<0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p=0.006). Conclusion: These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer. © 2013 Editrice Gastroenterologica Italiana S.r.l.