RTKN2 induces NF-KappaB dependent resistance to intrinsic apoptosis in HEK cells and REgulates BCL-2 genes in human CD4+ lymphocytes

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4+ T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNFα. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4+ T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4+ T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.

The effects of escitalopram on myocardial apoptosis and the expression of Bax and Bcl-2 during myocardial ischemia/reperfusion in a model of rats with depression

BackgroundMajor depressive disorder (MDD) is an independent risk factor for coronary heart disease (CHD), and influences the occurrence and prognosis of cardiovascular events. Although there is evidence that antidepressants may be cardioprotective after acute myocardial infarction (AMI) comorbid with MDD, the operative pathophysiological mechanisms remain unclear. Our aim was therefore to explore the molecular mechanisms of escitalopram on myocardial apoptosis and the expression of Bax and Bcl-2 in a rat model of depression during myocardial ischemia/reperfusion (I/R).MethodsRats were divided randomly into 3 groups (n = 8): D group (depression), DI/R group (depression with myocardial I/R) and escitalopram + DI/R group. The rats in all three groups underwent the same chronic mild stress and separation for 21 days, at the same time, in the escitalopram + DI/R group, rats were administered escitalopram by gavage (10 mg/kg/day). Ligation of the rat¿s left anterior descending branch was done in the myocardial I/R model. Following which behavioral tests were done. The size of the myocardial infarction was detected using 1.5% TTC dye. The Tunel method was used to detect apoptotic myocardial cells, and both the Rt-PCR method and immunohistochemical techniques were used to detect the expression of Bcl¿2 and Bax.ResultsCompared with the D and DI/R groups, rats in Escitalopram + DI/R group showed significantly increased movements and sucrose consumption (P < .01). Compared with the DI/R group, the myocardial infarct size in the escitalopram + DI/R group was significantly decreased (P < .01). Compared with the D group, there were significantly increased apoptotic myocardial cells in the DI/R and escitalopram + DI/R groups (P < .01); however compared with the DI/R group, apoptotic myocardial cell numbers in the escitalopram + DI/R group were significantly decreased (P < .01). Compared with the DI/R group, there was a down-regulated Bax:Bcl-2 ratio in the escitalopram + DI/R group (P < .01).ConclusionsThese results suggest that in patients with AMI comorbid with MDD, there is an increase in pro-apoptotic pathways that is reversed by escitalopram. This suggests that clinically escitalopram may have a direct cardioprotective after acute myocardial infarction.

NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - but not palmitate-induced toxicity

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

Graphene quantum dots induce apoptosis, autophagy, and inflammatory response via p38 mitogen-activated protein kinase and nuclear factor-κB mediated signaling pathways in activated THP-1 macrophages.

The biomedical application of graphene quantum dots (GQDs) is a new emerging area. However, their safety data are still in scarcity to date. Particularly, the effect of GQDs on the immune system remains unknown. This study aimed to elucidate the interaction of GQDs with macrophages and the underlying mechanisms. Our results showed that GQDs slightly affected the cell viability and membrane integrity of macrophages, whereas GQDs significantly increased reactive oxygen species (ROS) generation and apoptotic and autophagic cell death with an increase in the expression level of Bax, Bad, caspase 3, caspase 9, beclin 1, and LC3-I/II and a decrease in that of Bcl-2. Furthermore, low concentrations of GQDs significantly increased the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-8, whereas high concentrations of GQDs elicited opposite effects on the cytokines production. SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), abolished the cytokine-inducing effect of GQDs in macrophages. Moreover, GQDs significantly increased the phosphorylation of p38 MAPK and p65, and promoted the nuclear translocation of nuclear factor-κB (NF-κB). Taken together, these results show that GQDs induce ROS generation, apoptosis, autophagy, and inflammatory response via p38MAPK and NF-κB mediated signaling pathways in THP-1 activated macrophages.

Angiotensin II receptor imbalance associated with neonatal cardiac growth restriction is a prelude to adult cardiac hypertrophy

The Hypertrophic Heart Rat (HHR) displays spontaneous cardiomyocyte hypertrophy in association with an apparent reduction in myocyte number in adulthood. This suggests the possibility of reduced hyperplasia or increased apoptosis during early cardiac development. The angiotensin AT1 and AT2 receptor subtypes have been implicated in both cellular growth and apoptosis, but the precise mechanisms are unclear. The aim of this study was to determine the relationship between cardiac AngII receptor expression levels and neonatal cardiomyocyte growth and apoptotic responses in the HHR compared with the Normal Heart Rat (NHR) control strain. Cardiac tissues were freshly harvested from male HHR and NHR at several developmental stages (p2 and 4, 6, 8, 12wks). HHR cardiac weight indices were considerably smaller than NHR at day 2 (4.330.19 vs 5.010.08 mg/g), but ‘caught-up’ to NHR by 4 weeks (5.100.15 vs 5.160.11 mg/g). By 12 weeks, HHR hearts were 27% larger than NHR. Tissue AT1A and AT2 mRNA expression levels were quantified by real-time RT-PCR. Relative to NHR, HHR neonatal hearts exhibited a 4.6-fold higher AT2/AT1 mRNA expression ratio. Cultured neonatal cardiomyocytes were infected with AT1A and/or AT2 receptor-expressing adenoviruses to achieve a physiological level of receptor expression (150 fmol receptor protein/mg total cell protein). In addition, to emulate receptor expression in neonatal HHR hearts, cells were co-infected with AT1A and AT2 receptors at a 4:1 ratio. Apoptosis incidence was studied by morphological analysis after 72 hours exposure to 0.1 M AngII. When infected with the AT1A receptor alone, a higher proportion of HHR myocytes appeared apoptotic than NHR (22.7 4.1% vs 1.1 0.6%, P 0.001). This implies that intrinsic differences predispose HHR cells to accentuated AT1-mediated apoptosis. Interestingly, the bax-1/bcl-2 mRNA expression ratio was significantly higher (50%) in HHR neonatal hearts. When cells were co-infected with AT1A and AT2 receptors, evidence of apoptosis in HHR cells virtually disappeared (0.4 0.1%). These findings suggest a novel capacity of AT2 receptors to counteract accentuated AT1A receptor-induced apoptosis in the HHR in early cardiac growth.

Rhotekin 2, a novel GTPase effector involved in resistance to apoptosis

This thesis describes the discovery, characterization, and function of a new protein, Rhotekin 2. It is a signaling protein with a significant role in resistance to cell death in human lymphocytes and therefore may be a potential new target in future treatments of immune diseases such a HIV and leukemia.

Advanced graph mining methods for protein analysis

As one of the primary substances in a living organism, protein defines the character of each cell by interacting with the cellular environment to promote the cell’s growth and function [1]. Previous studies on proteomics indicate that the functions of different proteins could be assigned based upon protein structures [2,3]. The knowledge on protein structures gives us an overview of protein fold space and is helpful for the understanding of the evolutionary principles behind structure. By observing the architectures and topologies of the protein families, biological processes can be investigated more directly with much higher resolution and finer detail. For this reason, the analysis of protein, its structure and the interaction with the other materials is emerging as an important problem in bioinformatics. However, the determination of protein structures is experimentally expensive and time consuming, this makes scientists largely dependent on sequence rather than more general structure to infer the function of the protein at the present time. For this reason, data mining technology is introduced into this area to provide more efficient data processing and knowledge discovery approaches.

Unlike many data mining applications which lack available data, the protein structure determination problem and its interaction study, on the contrary, could utilize a vast amount of biologically relevant information on protein and its interaction, such as the protein data bank (PDB) [4], the structural classification of proteins (SCOP) databases [5], CATH databases [6], UniProt [7], and others. The difficulty of predicting protein structures, specially its 3D structures, and the interactions between proteins as shown in Figure 6.1, lies in the computational complexity of the data. Although a large number of approaches have been developed to determine the protein structures such as ab initio modelling [8], homology modelling [9] and threading [10], more efficient and reliable methods are still greatly needed.

In this chapter, we will introduce a state-of-the-art data mining technique, graph mining, which is good at defining and discovering interesting structural patterns in graphical data sets, and take advantage of its expressive power to study protein structures, including protein structure prediction and comparison, and protein-protein interaction (PPI). The current graph pattern mining methods will be described, and typical algorithms will be presented, together with their applications in the protein structure analysis.

The rest of the chapter is organized as follows: Section 6.2 will give a brief introduction of the fundamental knowledge of protein, the publicly accessible protein data resources and the current research status of protein analysis; in Section 6.3, we will pay attention to one of the state-of-the-art data mining methods, graph mining; then Section 6.4 surveys several existing work for protein structure analysis using advanced graph mining methods in the recent decade; finally, in Section 6.5, a conclusion with potential further work will be summarized.

The effects of apoptosis vulnerability markers on the myocardium in depression after myocardial infarction

Background : There is an increased incidence of major depressive disorder (MDD) in individuals after myocardial infarction (MI), but the pathophysiological processes mediating this association are unclear. Our previous study demonstrated an increase in pro-apoptotic pathways in the myocardium and hippocampus in MDD, which was reversed by venlafaxine. This study aimed to attempt to confirm the effects of apoptosis vulnerability markers on the myocardium in a model of depression after myocardial infarction.

Methods : Rats were divided into four groups: sham (N = 8), depression (N = 8, chronic mild unpredictable stress and separation were used in the depression group), MI (N = 13) and post-MI depression (N = 7). The rats in all four groups underwent the same open field and sucrose preference behavioral tests. Evan Blue staining was used to determine the area at risk of myocardial infarction in the left ventricle, and 2,3,5-triphenyl tetrazolium chloride (1.5% TTC) dye was used to detect the size of the myocardial infarction. The expression of bax and bcl-2 protein in the myocardium was investigated by immunohistochemistry, and the mRNA expression of bax, bcl-2 and caspase-3 in the myocardium was investigated by real time RT-PCR. Apoptosis was estimated in the myocardium by measuring the Bax:Bcl-2 ratio.

Results : In the depression and post-MI depression rats, there were significantly decreased movements and total sucrose consumption, modeling behavioral deficits and an anhedonic-like state. In terms of myocardial infarction size, no difference was seen between the MI and post-MI depression groups. There was an up-regulated Bax:Bcl-2 ratio in the depression, MI and post-MI depression groups. Furthermore, in the latter group, there was a greater up-regulated Bax:Bcl-2 ratio. However, caspase-3 did not differ among the four groups.

Conclusions : These results of this animal model suggest that active pro-apoptotic pathways may be involved in the nexus between myocardial infarction and depression. This mechanism may be germane to understanding this relationship in humans.

Synergistic effects of IAP inhibitor LCL161 and paclitaxel on hepatocellular carcinoma cells

Inhibitor of Apoptosis Proteins (IAPs) are key regulators of apoptosis in hepatocellular carcinoma (HCC) and their expression is negatively correlated with patient survival. LCL161 is a small molecule inhibitor of IAPs that has potent antitumour activity in a range of solid tumours. In HCC, response to LCL161 therapy has shown to be mediated by Bcl-2 expression. In this study, we aim to determine whether LCL161 has any therapeutic potential in HCC. Protein expression was determined by Western blot. Cell proliferation was determined by Cell Proliferation ELISA and BrdU colorimetric assays. Apoptosis was determined by Annexin V assay. Cell cycle analysis was performed by staining cells with propidium iodide and analysed in a FACScan. Automated Cell Counter and phase contrast microscopy were used to determine the cell viability. We have found that LCL161 targets (cIAP1, cIAP2 and XIAP) were up-regulated in HCC tumours. Both high Bcl-2 expressing HuH7 cells and low Bcl-2 expressing SNU423 cells showed strong resistance to LCL161 therapy with significant effects on both apoptosis and cell viability only evident at LCL161 concentrations of ⩾100μM. At these doses there was significant inhibition of IAP targets, however there was also significant inhibition of off-target proteins including pERK and pJNK suggesting apoptosis caused by drug toxicity. However, when used in combination with paclitaxel in HuH7 and SNU423 cells, LCL161 had significant antiproliferative effects at doses as low as 2μM and this was independent of Bcl-2 expression. Thus, LCL161 may be a useful agent in combination with paclitaxel to treat liver tumours.

Smoothened activates breast cancer stem-like cell and promotes tumorigenesis and metastasis of breast cancer

Smoothened (Smo) is a G protein-coupled receptor protein encoded by the Smo gene of the hedgehog signalling pathway, which is thought to play an important role in maintaining organ patterning, cell differentiation and self-renewal. The possible role of Smo in the process of tumorigenesis and metastasis of breast cancer still remains unclear. The present experiments were to investigate the effect of Smo on activating breast cancer stem-like CD44(+)CD24(-) cells and the tumorigenesis and metastasis of breast cancer. By injected CD44(+)CD24(-) cells (1×10(4)) into the cleared fat pad of NOD/SCID mice, it was observed that CD44(+)CD24(-) cells possess higher tumor-initiating capacity and metastasis properties than equal numbers of non-CD44(+)CD24(-) cells. The mRNA and protein expressions of Smo in CD44(+)CD24(-) cells were higher than those in non-CD44(+)CD24(-) cells, indicating that Smo may play a role in maintaining breast cancer stem cell features. qRT-PCR results revealed that expressions of STAT3, Bcl-2 and cyclinD1 mRNA in MCF-7 cells were decreased after transfected by Smo siRNA. In addition, the expressions of MMP-2 and MMP-9 were downregulated in MCF-7 cells after Smo expression was inhibited. Smo inhibition may be a possible therapeutic target that potentially suppresses breast tumor formation and development.

Bardoxolone methyl induces apoptosis and autophagy and inhibits epithelial-to-mesenchymal transition and stemness in esophageal squamous cancer cells

Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me) on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC) cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9, and cleaved poly ADP ribose polymerase (PARP) levels but increased the expression level of Bcl-2-associated X (Bax). Furthermore, CDDO-Me induced autophagy in both Ec109 and KYSE70 cells via suppression of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. There were interactions between the autophagic and apoptotic pathways in Ec109 and KYSE70 cells subject to CDDO-Me treatment. CDDO-Me also scavenged reactive oxygen species through activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) pathway in Ec109 and KYSE70 cells. CDDO-Me inhibited cell invasion, epithelial-mesenchymal transition, and stemness in Ec109 and KYSE70 cells. CDDO-Me significantly downregulated E-cadherin but upregulated Snail, Slug, and zinc finger E-box-binding homeobox 1 (TCF-8/ZEB1) in Ec109 and KYSE70 cells. CDDO-Me significantly decreased the expression of octamer-4, sex determining region Y-box 2 (Sox-2), Nanog, and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), all markers of cancer cell stemness, in Ec109 and KYSE70 cells. Taken together, these results indicate that CDDO-Me is a promising anticancer agent against ESCC. Further studies are warranted to explore the molecular targets, efficacy and safety of CDDO-Me in the treatment of ESCC.

The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and β-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy.

Regionally specific changes in levels of cortical S100β in bipolar 1 disorder but not schizophrenia

Objective: To determine if levels of the glial-derived proteins S100β and glial acidic fibrillary protein (GFAP) and the pro- and antiapoptotic proteins p53 and Bcl-2 were altered in the cortex of subjects with schizophrenia or bipolar 1 disorder.
Method: Levels of S100β, GFAP, p53 and Bcl-2 were measured in cortex (Brodmann's Areas (BAs) 9, 10, 46 and 40) of control subjects and subjects with schizophrenia, bipolar 1 disorder and in the cortex of rats treated with haloperidol or lithium using protein-specific antibodies and western blot analysis.
Results: Levels of S100β were decreased in BA 9 and increased in BA 40 from subjects with bipolar 1 disorder. Levels of this protein were not altered in other CNS regions, in schizophrenia or in the cortex of rats treated with haloperidol or lithium. No changes in levels of the other three proteins were detected across diagnoses.
Conclusions: Regionally selective changes in cortical S100β may be associated with the pathology of bipolar 1 disorder and may reflect derangements in neuronal death or survival

Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer

BACKGROUND: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities. METHODS: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin. RESULTS: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells. CONCLUSIONS: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.