Mechanisms of doxorubicin-induced drug resistance and drug resistant tumour growth in a murine breast tumour model.

BACKGROUND: Doxorubicin is currently the most effective chemotherapeutic drug used to treat breast cancer. It has, however, been shown that doxorubicin can induce drug resistance resulting in poor patient prognosis and survival. Studies reported that the interaction between signalling pathways can promote drug resistance through the induction of proliferation, cell cycle progression and prevention of apoptosis. The aim of this study was therefore to determine the effects of doxorubicin on apoptosis signalling, autophagy, the mitogen-activated protein kinase (MAPK)- and phosphoinositide 3-kinase (PI3K)/Akt signalling pathway, cell cycle control, and regulators of the epithelial-mesenchymal transition (EMT) process in murine breast cancer tumours. METHODS: A tumour-bearing mouse model was established by injecting murine E0771 breast cancer cells, suspended in Hank's Balances Salt Solution and Corning® Matrigel® Basement Membrane Matrix, into female C57BL/6 mice. Fourty-seven mice were randomly divided into three groups, namely tumour control (received Hank's Balances Salt Solution), low dose doxorubicin (received total of 6 mg/ml doxorubicin) and high dose doxorubicin (received total of 15 mg/ml doxorubicin) groups. A higher tumour growth rate was, however, observed in doxorubicin-treated mice compared to the untreated controls. We therefore compared the expression levels of markers involved in cell death and survival signalling pathways, by means of western blotting and fluorescence-based immunohistochemistry. RESULTS: Doxorubicin failed to induce cell death, by means of apoptosis or autophagy, and cell cycle arrest, indicating the occurrence of drug resistance and uncontrolled proliferation. Activation of the MAPK/ extracellular-signal-regulated kinase (ERK) pathway contributed to the resistance observed in treated mice, while no significant changes were found with the PI3K/Akt pathway and other MAPK pathways. Significant changes were also observed in cell cycle p21 and DNA replication minichromosome maintenance 2 proteins. No significant changes in EMT markers were observed after doxorubicin treatment. CONCLUSIONS: Our results suggest that doxorubicin-induced drug resistance and tumour growth can occur through the adaptive role of the MAPK/ERK pathway in an effort to protect tumour cells. Previous studies have shown that the efficacy of doxorubicin can be improved by inhibition of the ERK signalling pathway and thereby treatment failure can be overcome.

Cancer stem cells: A product of clonal evolution?

The cancer stem cell (CSC) model has emerged as a prominent paradigm for explaining tumour heterogeneity. CSCs in tumour recurrence and drug resistance have also been implicated in a number of studies. In fact, CSCs are often identified by their expression of drug-efflux proteins which are also highly expressed in normal stem cells. Similarly, pro-survival or proliferation signalling often exhibited by stem cells is regularly reported as being upregulated by CSC. Here we review evidence suggesting that many aspects of CSCs are more readily described by clonal evolution. As an example, cancer cells often exhibit copy number gains of genes involved in drug-efflux proteins and pro-survival signalling. Consequently, clonal selection for stem cell traits may result in cancer cells developing "stemness" traits which impart a fitness advantage, without strictly following a CSC model. Finally, since symmetric cell division would give rise to more cells than asymmetric division, it is expected that more advanced tumours would depart from a CSC. Collectively, these observations suggest clonal evolution may explain many aspects of the CSC.

Enhanced Therapeutic Efficacy in Cancer Patients by Short-term Fasting: The Autophagy Connection.

Preclinical studies suggest that fasting prior to chemotherapy may be an effective strategy to protect patients against the adverse effects of chemo-toxicity. Fasting may also sensitize cancer cells to chemotherapy. It is further suggested that fasting may similarly augment the efficacy of oncolytic viral therapy. The primary mechanism mediating these beneficial effects is thought to relate to the fact that fasting results in a decrease of circulating growth factors. In turn, such fasting cues would prompt normal cells to redirect energy toward cell maintenance and repair processes, rather than growth and proliferation. However, fasting is also known to upregulate autophagy, an evolutionarily conserved catabolic process that is upregulated in response to various cell stressors. Here, we review a number of mechanisms by which fasting-induced autophagy may have an impact on both chemo-tolerance and chemo-sensitization. First, fasting may exert a protective effect by mobilizing autophagic components prior to chemo-induction. In turn, the autophagic apparatus can be repurposed for removing cellular components damaged by chemotherapy. Autophagy also plays a key role in epitope expression as well as in modulating inflammation. Chemo-sensitization resulting from fasting may in fact be an effect of enhanced immune surveillance as a result of better autophagy-dependent epitope processing. Finally, autophagy is involved in host defense against viruses, and aspects of the autophagic process are also often targets for viral subversion. Consequently, altering autophagic flux by fasting may alter viral infectivity. These observations suggest that fasting-induced autophagy may have an impact on therapeutic efficacy in various oncological contexts.

A proteomic analysis of the ventral hippocampus of rats subjected to maternal separation and escitalopram treatment.

Early life stress is known to predispose humans to the development of depression. Developmental stress has been shown to cause various changes in neurotransmitter systems, neurotrophin expression and the hypothalamic pituitary adrenal-axis in the rat brain. The aim of this study was to identify which cytosolic proteins are altered by maternal separation, as a model for depression, as well as by chronic antidepressant treatment. Rats were maternally separated from postnatal day 2-14 for 3 h per day while control rats were normally reared. Both groups were divided and received either escitalopram or saline injections for 6 weeks starting from postnatal day 40. The ventral hippocampal tissue was fractionated and the cytosolic fraction used for 2-D-gel electrophoresis and liquid chromatography coupled to mass spectrometry analyses to identify peptides. Mascot database searches were done to identify proteins that were differentially expressed between the groups. Proteins that were significantly changed by maternal separation included amongst others: molecular chaperones and proteins related to energy metabolism; neuroplasticity; oxidative stress regulation; and protein metabolism. Treatment with escitalopram, a selective-serotonin reuptake inhibitor, induced changes in a different group of proteins, except for a few involved in energy metabolism and neuroprotective pathways. The results indicate which cytosolic proteins are changed by early life stress and may therefore be involved in the development of depression.

Behavioral and quantitative mitochondrial proteome analyses of the effects of simvastatin: implications for models of neural degeneration.

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, is used for lowering elevated low-density lipoprotein cholesterol concentrations. This translates into reduced cardiovascular disease-related morbidity and mortality, while the drugs' anti-oxidant and anti-inflammatory properties have earmarked it as a potential treatment strategy against various neurological conditions. Statins have been shown to protect neurons from degeneration in a number of animal models. Although no mechanism completely explains the multiple benefits exerted by statins, emerging evidence suggests that in some degenerative and brain injury models, mitochondrial impairment may play a contributive rate. However, [corrected] evidence lacks to support a directly influencing role for statins on mitochondria-related proteins and motor behavior. Mitochondrial dysfunction may increase oxygen free radical production, which in turn leaves cells susceptible to energy failure, apoptosis and related events [corrected] which could prove fatal. The potential link between simvastatin treatment and mitochondrial function would be supported if key mitochondrial proteins were altered by simvastatin exposure. Using mass spectroscopy (MS), we identified 24 mitochondrial proteins that differed significantly (P < 0.05) in relative abundancy as a result of simvastatin treatment. The identified proteins represented many facets of mitochondrial integrity, with the majority forming part of the electron transport chain machinery, which is necessary for energy production. In a follow-up study, we then addressed whether simvastatin is capable of altering sensorimotor function in a mitochondrial toxin-induced animal model. Rats were pre-treated with simvastatin for 14 days, followed by a single unihemispheric (substantia nigra; SN) injection of rotenone, a mitochondrial complex I (Co-I) inhibitor. Results showed that simvastatin improved motor performance in rotenone-infused rats. The data are consistent with the possibility that alteration of mitochondrial function may contribute to the beneficial effects associated with statin use.

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway.

UNLABELLED: The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. OBJECTIVE: To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. METHODS: Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO(2) incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. RESULTS: (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. CONCLUSIONS: Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.

Large scale hippocampal cellular distress may explain the behavioral consequences of repetitive traumatic experiences--a proteomic approach.

Early life traumatic experiences are associated with psychopathology in adulthood. This may be due in part to the effects of trauma on hippocampal development and protein expression. The purpose of the study was to investigate the effects of early life trauma and adult re-stress on ventral hippocampal protein expression. Adolescent rats (n = 19) were subjected to a triple stressor on post-natal day 28 followed 7 days later by the first re-stress session and 25 days later (post-natal day 60 = adulthood) by the second re-stress session. Ventral hippocampi were collected on post-natal day 68 for protein expression determinations using protein arrays and 2D-gel electrophoresis with liquid chromatography tandem mass spectrometry. Compared to controls, traumatized animals showed an increase in Ca(2+) homeostatic proteins, dysregulated signaling pathways and energy metabolism enzymes, cytoskeletal protein changes, a decrease in neuroplasticity regulators, energy metabolism enzymes and an increase in apoptotic initiator proteins. These results indicate the extensive impact of trauma on adult brain development and behavior.

Nitric oxide production is higher in rat cardiac microvessel endothelial cells than ventricular cardiomyocytes in baseline and hypoxic conditions: a comparative study.

UNLABELLED: The relative importance of endothelium- and cardiomyocyte-derived nitric oxide (NO) is unknown, with a lack of direct studies on cardiac microvessel endothelial cells (CMEC) and cardiomyocytes regarding relative cellular NO production. AIMS: To assess and compare baseline and hypoxia-induced NO and ONOO- production in cardiomyocytes and CMEC. METHODS: Rat cardiomyocytes were isolated, and cultured rat CMEC were purchased commercially. Hypoxia (+/- NOS inhibitors) was induced by mineral oil layering or hypoxic culture. NO and ONOO- were detected by FACS analysis of DAF-2/DA and DHR123, respectively. Total eNOS was determined by Western blot analysis. RESULTS: 1) Baseline NO production in CMEC was sevenfold (cultured cells) and 26-fold (isolated cells) higher than in cardiomyocytes, 2) eNOS expression was 22-fold higher in CMEC, 3) hypoxia increased NO production in both cell types, albeit to a larger extent in CMEC, 4) in hypoxic cardiomyocytes, nonselective NOS and iNOS-specific inhibition attenuated NO production, whereas in CMEC, iNOS-specific inhibition was ineffective, and 5) baseline ONOO- production was 2.2 times greater in CMEC than in cardiomyocytes. CONCLUSION: Using a novel approach, this study demonstrated that CMEC produce more baseline NO than cardiomyocytes, and that hypoxia activates NOS to increase NO production in both cell types. Baseline eNOS content was higher in CMEC than in cardiomyocytes, suggesting that differences in baseline NO production were eNOS-associated.