Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

Lattice-free descriptions of collective motion with crowding and adhesion

Cell-to-cell adhesion is an important aspect of malignant spreading that is often observed in images from the experimental cell biology literature. Since cell-to-cell adhesion plays an important role in controlling the movement of individual malignant cells, it is likely that cell-to-cell adhesion also influences the spatial spreading of populations of such cells. Therefore, it is important for us to develop biologically realistic simulation tools that can mimic the key features of such collective spreading processes to improve our understanding of how cell-to-cell adhesion influences the spreading of cell populations. Previous models of collective cell spreading with adhesion have used lattice-based random walk frameworks which may lead to unrealistic results, since the agents in the random walk simulations always move across an artificial underlying lattice structure. This is particularly problematic in high-density regions where it is clear that agents in the random walk align along the underlying lattice, whereas no such regular alignment is ever observed experimentally. To address these limitations, we present a lattice-free model of collective cell migration that explicitly incorporates crowding and adhesion. We derive a partial differential equation description of the discrete process and show that averaged simulation results compare very well with numerical solutions of the partial differential equation.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

RanGTPase : a candidate for myc-mediated cancer progression

Background Ras-related nuclear protein (Ran) is required for cancer cell survival in vitro and human cancer progression, but the molecular mechanisms are largely unknown. Methods We investigated the effect of the v-myc myelocytomatosis viral oncogene homolog (Myc) on Ran expression by Western blot, chromatin immunoprecipitation, and luciferase reporter assays and the effects of Myc and Ran expression in cancer cells by soft-agar, cell adhesion, and invasion assays. The correlation between Myc and Ran and the association with patient survival were investigated in 14 independent patient cohorts (n = 2430) and analyzed with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. Results Myc binds to the upstream sequence of Ran and transactivates Ran promoter activity. Overexpression of Myc upregulates Ran expression, whereas knockdown of Myc downregulates Ran expression. Myc or Ran overexpression in breast cancer cells is associated with cancer progression and metastasis. Knockdown of Ran reverses the effect induced by Myc overexpression in breast cancer cells. In clinical data, a positive association between Myc and Ran expression was revealed in 288 breast cancer and 102 lung cancer specimens. Moreover, Ran expression levels differentiate better or poorer survival in Myc overexpressing breast (χ2 = 24.1; relative risk [RR] = 9.1, 95% confidence interval [CI] = 3.3 to 24.7, P <. 001) and lung (χ2 = 6.04; RR = 2.8, 95% CI = 1.2 to 6.3; P =. 01) cancer cohorts. Conclusions Our results suggest that Ran is required for and is a potential therapeutic target of Myc-driven cancer progression in both breast and lung cancers. © 2013 The Author.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

Recasting the theory of mosquito-borne pathogen transmission dynamics and control

Mosquito-borne diseases pose some of the greatest challenges in public health, especially in tropical and sub-tropical regions of theworld. Efforts to control these diseases have been underpinned by a theoretical framework developed for malaria by Ross and Macdonald, including models, metrics for measuring transmission, and theory of control that identifies key vulnerabilities in the transmission cycle. That framework, especially Macdonald’s formula for R0 and its entomological derivative, vectorial capacity, are nowused to study dynamics and design interventions for many mosquito-borne diseases. A systematic review of 388 models published between 1970 and 2010 found that the vast majority adopted the Ross–Macdonald assumption of homogeneous transmission in a well-mixed population. Studies comparing models and data question these assumptions and point to the capacity to model heterogeneous, focal transmission as the most important but relatively unexplored component in current theory. Fine-scale heterogeneity causes transmission dynamics to be nonlinear, and poses problems for modeling, epidemiology and measurement. Novel mathematical approaches show how heterogeneity arises from the biology and the landscape on which the processes of mosquito biting and pathogen transmission unfold. Emerging theory focuses attention on the ecological and social context formosquito blood feeding, themovement of both hosts and mosquitoes, and the relevant spatial scales for measuring transmission and for modeling dynamics and control.

How much information can be obtained from tracking the position of the leading edge in a scratch assay?

Moving cell fronts are an essential feature of wound healing, development and disease. The rate at which a cell front moves is driven, in part, by the cell motility, quantified in terms of the cell diffusivity $D$, and the cell proliferation rate �$\lambda$. Scratch assays are a commonly-reported procedure used to investigate the motion of cell fronts where an initial cell monolayer is scratched and the motion of the front is monitored over a short period of time, often less than 24 hours. The simplest way of quantifying a scratch assay is to monitor the progression of the leading edge. Leading edge data is very convenient since, unlike other methods, it is nondestructive and does not require labeling, tracking or counting individual cells amongst the population. In this work we study short time leading edge data in a scratch assay using a discrete mathematical model and automated image analysis with the aim of investigating whether such data allows us to reliably identify $D$ and $\lambda$�. Using a naıve calibration approach where we simply scan the relevant region of the ($D$;$\lambda$�) parameter space, we show that there are many choices of $D$ and $\lambda$� for which our model produces indistinguishable short time leading edge data. Therefore, without due care, it is impossible to estimate $D$ and $\lambda$� from this kind of data. To address this, we present a modified approach accounting for the fact that cell motility occurs over a much shorter time scale than proliferation. Using this information we divide the duration of the experiment into two periods, and we estimate $D$ using data from the first period, while we estimate �$\lambda$ using data from the second period. We confirm the accuracy of our approach using in silico data and a new set of in vitro data, which shows that our method recovers estimates of $D$ and $\lamdba$� that are consistent with previously-reported values except that that our approach is fast, inexpensive, nondestructive and avoids the need for cell labeling and cell counting.

Understanding the information literacy experiences of EFL (English as a Foreign Language) students

This thesis investigated the information literacy experiences of EFL (English as a Foreign Language) students in a higher education institution in the United Arab Emirates (UAE). Phenomenography was used to investigate how EFL students' 'used information to learn' (ie. information literacy). The study revealed that EFL students' experienced information literacy across four categories and had varying experiences of information and learning. The research also showed that EFL students' faced a number of challenges and barriers due to language that impacted on their experiences of reading, understanding, accessing and translating information.

The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening

Fruit softening in apple (Malus 3 domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A coldrelated gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the coldand ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

Interaction of psychosocial risk factors explain increased neck problems among female office workers

This study investigated the relationship between psychosocial risk factors and (1) neck symptoms and (2) neck pain and disability as measured by the neck disability index (NDI). Female office workers employed in local private and public organizations were invited to participate, with 333 completing a questionnaire. Data were collected on various risk factors including age, negative affectivity, history of previous neck trauma, physical work environment, and task demands. Sixty-one percent of the sample reported neck symptoms lasting greater than 8 days in the last 12 months. The mean NDI of the sample was 15.5 out of 100, indicating mild neck pain and disability. In a hierarchical multivariate logistic regression, low supervisor support was the only psychosocial risk factor identified with the presence of neck symptoms. Similarly, low supervisor support was the only factor associated with the score on the NDI. These associations remained after adjustment for potential confounders of age, negative affectivity, and physical risk factors. The interaction of job demands, decision authority, and supervisor support was significantly associated with the NDI in the final model and this association increased when those with previous trauma were excluded. Interestingly, and somewhat contrary to initial expectations, as job demands increased, high decision authority had an increasing effect on the NDI when supervisor support was low.

Quantitative sensory measures distinguish office workers with varying levels of neck pain and disability

This study was undertaken to investigate any relationship between sensory features and neck pain in female office workers using quantitative sensory measures to better understand neck pain in this group. Office workers who used a visual display monitor for more than four hours per day with varying levels of neck pain and disability were eligible for inclusion. There were 85 participants categorized according to their scores on the neck disability index (NDI): 33 with no pain (NDI < 8); 38 with mild levels of pain and disability (NDI 9–29); 14 with moderate levels of pain (NDI ⩾ 30). A fourth group of women without neck pain (n = 22) who did not work formed the control group. Measures included: thermal pain thresholds over the posterior cervical spine; pressure pain thresholds over the posterior neck, trapezius, levator scapulae and tibialis anterior muscles, and the median nerve trunk; sensitivity to vibrotactile stimulus over areas of the hand innervated by the median, ulnar and radial nerves; sympathetic vasoconstrictor response. All tests were conducted bilaterally. ANCOVA models were used to determine group differences between the means for each sensory measure. Office workers with greater self-reported neck pain demonstrated hyperalgesia to thermal stimuli over the neck, hyperalgesia to pressure stimulation over several sites tested; hypoaesthesia to vibration stimulation but no changes in the sympathetic vasoconstrictor response. There is evidence of multiple peripheral nerve dysfunction with widespread sensitivity most likely due to altered central nociceptive processing initiated and sustained by nociceptive input from the periphery.

Alterations in cervical muscle activity in functional and stressful tasks in female office workers with neck pain

This study determined differences between computer workers with varying levels of neck pain in terms of work stressors, employee strain, electromyography (EMG) amplitude and heart rate response to various tasks. Participants included 85 workers (33, no pain; 38, mild pain; 14, moderate pain) and 22 non-working controls. Work stressors evaluated were job demands, decision authority, and social support. Heart rate was recorded during three tasks: copy-typing, typing with superimposed stress and a colour word task. Measures included electromyography signals from the sternocleidomastoid (SCM), anterior scalene (AS), cervical extensor (CE) and upper trapezius (UT) muscles bilaterally. Results showed no difference between groups in work stressors or employee strain measures. Workers with and without pain had higher measured levels of EMG amplitude in SCM, AS and CE muscles during the tasks than controls (all P < 0.02). In workers with neck pain, the UT had difficulty in switching off on completion of tasks compared with controls and workers without pain. There was an increase in heart rate, perceived tension and pain and decrease in accuracy for all groups during the stressful tasks with symptomatic workers producing more typing errors than controls and workers without pain. These findings suggest an altered muscle recruitment pattern in the neck flexor and extensor muscles. Whether this is a consequence or source of the musculoskeletal disorder cannot be determined from this study. It is possible that workers currently without symptoms may be at risk of developing a musculoskeletal disorder.

Neck movement and muscle activity characteristics in female office workers with neck pain

Study Design Cross-sectional study. Objective To explore aspects of cervical musculoskeletal function in female office workers with neck pain. Summary of Background Data Evidence of physical characteristics that differentiate computer workers with and without neck pain is sparse. Patients with chronic neck pain demonstrate reduced motion and altered patterns of muscle control in the cervical flexor and upper trapezius (UT) muscles during specific tasks. Understanding cervical musculoskeletal function in office workers will better direct intervention and prevention strategies. Methods Measures included neck range of motion; superficial neck flexor muscle activity during a clinical test, the craniocerivcal flexion test; and a motor task, a unilateral muscle coordination task, to assess the activity of both the anterior and posterior neck muscles. Office workers with and without neck pain were formed into 3 groups based on their scores on the Neck Disability Index. Nonworking women without neck pain formed the control group. Surface electromyographic activity was recorded bilaterally from the sternocleidomastoid, anterior scalene (AS), cervical extensor (CE) and UT muscles. Results Workers with neck pain had reduced rotation range and increased activity of the superficial cervical flexors during the craniocervical flexion test. During the coordination task, workers with pain demonstrated greater activity in the CE muscles bilaterally. On completion of the task, the UT and dominant CE and AS muscles demonstrated an inability to relax in workers with pain. In general, there was a linear relationship between the workers’ self-reported levels of pain and disability and the movement and muscle changes. Conclusion These results are consistent with those found in other cervical musculoskeletal disorders and may represent an altered muscle recruitment strategy to stabilize the head and neck. An exercise program including motor reeducation may assist in the management of neck pain in office workers.

Associations between individual and workplace risk factors for self-reported neck pain and disability among female office workers

A cross-sectional survey of female office workers (n=333) was undertaken to determine the level of neck pain and disability (Neck Disability Index—NDI) and to explore the relationship between individual and workplace risk factors with the NDI score and the presence of pain. Workers reported nil (32%), mild (53%), moderate (14%) and severe (1%) neck pain. There were more risk factors associated with the NDI score than the presence of neck pain. The presence of neck pain was associated with a history of neck trauma (OR: 4.8), using a graduated lens (OR: 4.6), and negative affectivity (OR: 2.7) in the multiple regression model. Factors associated with higher NDI score were using the computer mouse for more than 6 h per day, higher negative affectivity, older age and an uncomfortable workstation. These results suggest that measuring the level of neck pain and disability rather than just the presence of neck pain provides more specific directives for the prevention and management of this disorder.