Nonlinear Feedback Excitation for System Interrogation by Bifurcation Morphing

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/76971/1/AIAA-35028-143.pd

Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells

<p>Abstract</p> <p>Background</p> <p>Chloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells.</p> <p>Methods</p> <p>HT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed.</p> <p>Results</p> <p>5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21^Cip1and p27^Kip1and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of 5-FU.</p> <p>Conclusion</p> <p>Our findings suggest that the combination therapy with CQ should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy, possibly by inhibiting autophagy-dependent resistance to chemotherapy.</p

Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

<p>Abstract</p> <p>Background</p> <p><it>ARHI </it>is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma <it>in situ </it>(DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel.</p> <p>Methods</p> <p>Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts.</p> <p>Results</p> <p>ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest.</p> <p>Conclusions</p> <p>ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.</p

Nurse staffing, medical staffing and mortality in intensive care: an observational study

Objectives: To investigate whether the size of the workforce (nurses, doctors and support staff) has an impact of the survival chances of critically ill patients both in the intensive care unit (ICU) and in the hospital. Background: Investigations of intensive care outcomes suggest that some of the variation in patient survival rates might be related to staffing levels and workload, but the evidence is still equivocal. Data: Information about patients, including the outcome of care (whether the patient lived or died) came from the Intensive Care National Audit & Research Centre (ICNARC) Case Mix Programme. An Audit Commission survey of ICUs conducted in 1998 gave information about staffing levels. The merged dataset had information on 65 ICUs and 38,168 patients. This is currently the best available dataset for testing the relationship between staffing and outcomes in UK ICUs Design: A cross-sectional, retrospective, risk adjusted observational study. Methods: Multivariable, multilevel logistic regression. Outcome Measures: ICU and in-hospital mortality. Results: After controlling for patient characteristics and workload we found that higher numbers of nurses per bed and higher numbers of consultants were associated with higher survival rates. Further exploration revealed that the number of nurses had the greatest impact on patients at high risk of death whereas the effect of medical staffing was across the range of patient acuity. No relationship between patient outcomes and the number of support staff was found. Distinguishing between direct care and supernumerary nurses and restricting the analysis to patients who had been in the unit for more than 8 hours made little difference to the results. Separate analysis of in-unit and in-hospital survival showed that the clinical workforce in intensive care had a greater impact on ICU mortality than on hospital mortality which gives the study additional credibility. Conclusion: This study supports claims that the availability of medical and nursing staff is associated with the survival of critically ill patients and suggests that future studies should focus on the resources of the health care team. The results emphasise the urgent need for a prospective study of staffing levels and the organisation of care in ICUs

Concurrent MEK2 Mutation and BRAF Amplification Confer Resistance to BRAF and MEK Inhibitors in Melanoma

SummaryAlthough BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it

Benzyl Isothiocyanate Causes FoxO1-Mediated Autophagic Death in Human Breast Cancer Cells

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl--cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition