A Novel Microtubule Inhibitor 4SC-207 with Anti-Proliferative Activity in Taxane-Resistant Cells

Microtubule inhibitors are invaluable tools in cancer chemotherapy: taxanes and vinca alkaloids have been successfully used in the clinic over the past thirty years against a broad range of tumors. However, two factors have limited the effectiveness of microtubule inhibitors: toxicity and resistance. In particular, the latter is highly unpredictable, variable from patient to patient and is believed to be the cause of treatment failure in most cases of metastatic cancers. For these reasons, there is an increasing demand for new microtubule inhibitors that can overcome resistance mechanisms and that, at the same time, have reduced side effects. Here we present a novel microtubule inhibitor, 4SC-207, which shows strong anti-proliferative activity in a large panel of tumor cell lines with an average GI₅₀ of 11nM. In particular, 4SC-207 is active in multi-drug resistant cell lines, such as HCT-15 and ACHN, suggesting that it is a poor substrate for drug efflux pumps. 4SC-207 inhibits microtubule growth in vitro and in vivo and promotes, in a dose dependent manner, a mitotic delay/arrest, followed by apoptosis or aberrant divisions due to chromosome alignment defects and formation of multi-polar spindles. Furthermore, preliminary data from preclinical studies suggest low propensity towards bone marrow toxicities at concentrations that inhibit tumor growth in paclitaxel-resistant xenograft models. In summary, our results suggest that 4SC-207 may be a potential anti-cancer agent.     

Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background

An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods

Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results

Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion

We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.     

Microbial Environment Affects Innate Immunity in Two Closely Related Earthworm Species Eisenia andrei and Eisenia fetida

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.     

Angiogenic Properties of Human Dental Pulp Stem Cells

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.     

Preliminary Evidence That Anodal Transcranial Direct Current Stimulation Enhances Time to Task Failure of a Sustained Submaximal Contraction

The purpose of this study was to determine whether anodal transcranial direct current stimulation (tDCS) delivered while performing a sustained submaximal contraction would increase time to task failure (TTF) compared to sham stimulation. Healthy volunteers (n = 18) performed two fatiguing contractions at 20% of maximum strength with the elbow flexors on separate occasions. During fatigue task performance, either anodal or sham stimulation was delivered to the motor cortex for up to 20 minutes. Transcranial magnetic stimulation (TMS) was used to assess changes in cortical excitability during stimulation. There was no systematic effect of the anodal tDCS stimulation on TTF for the entire subject set (n = 18; p = 0.64). Accordingly, a posteriori subjects were divided into two tDCS-time groups: Full-Time (n = 8), where TTF occurred prior to the termination of tDCS, and Part-Time (n = 10), where TTF extended after tDCS terminated. The TTF for the Full-Time group was 31% longer with anodal tDCS compared to sham (p = 0.04), whereas TTF for the Part-Time group did not differ (p = 0.81). Therefore, the remainder of our analysis addressed the Full-Time group. With anodal tDCS, the amount of muscle fatigue was 6% greater at task failure (p = 0.05) and the amount of time the Full-Time group performed the task at an RPE between 8–10 (“very hard”) increased by 38% (p = 0.04) compared to sham. There was no difference in measures of cortical excitability between stimulation conditions (p = 0.90). That the targeted delivery of anodal tDCS during task performance both increased TTF and the amount of muscle fatigue in a subset of subjects suggests that augmenting cortical excitability with tDCS enhanced descending drive to the spinal motorpool to recruit more motor units. The results also suggest that the application of tDCS during performance of fatiguing activity has the potential to bolster the capacity to exercise under conditions required to derive benefits due to overload.     

MRI Plaque Imaging Detects Carotid Plaques with a High Risk for Future Cerebrovascular Events in Asymptomatic Patients

Purpose

The aim of this study was to investigate prospectively whether MRI plaque imaging can identify patients with asymptomatic carotid artery stenosis who have an increased risk for future cerebral events. MRI plaque imaging allows categorization of carotid stenosis into different lesion types (I–VIII). Within these lesion types, lesion types IV–V and VI are regarded as rupture-prone plaques, whereas the other lesion types represent stable ones.

Methods

Eighty-three consecutive patients (45 male (54.2%); age 54–88 years (mean 73.2 years)) presenting with an asymptomatic carotid stenosis of 50–99% according to ECST-criteria were recruited. Patients were imaged with a 1.5-T scanner. T1-, T2-, time-of-flight-, and proton-density weighted studies were performed. The carotid plaques were classified as lesion type I–VIII. Clinical endpoints were ischemic stroke, TIA or amaurosis fugax. Survival analysis and log rank test were used to ascertain statistical significance.

Results

Six out of 83 patients (7.2%) were excluded: 4 patients had insufficient MR image quality; 1 patient was lost-to-follow-up; 1 patient died shortly after the baseline MRI plaque imaging. The following results were obtained by analyzing the remaining 77 patients. The mean time of follow-up was 41.1 months.During follow-up, n = 9 (11.7%) ipsilateral ischemic cerebrovascular events occurred. Only patients presenting with the high-risk lesion types IV–V and VI developed an ipsilateral cerebrovascular event versus none of the patients presenting with the stable lesion types III, VII, and VIII (n = 9 (11.7%) vs. n = 0 (0%) during follow-up). Event-free survival was higher among patients with the MRI-defined stable lesion types (III, VII, and VIII) than in patients with the high-risk lesion types (IV–V and VI) (log rank test P<0.0001).

Conclusions

MRI plaque imaging has the potential to identify patients with asymptomatic carotid stenosis who are particularly at risk of developing future cerebral ischemia. MRI could improve selection criteria for invasive therapy in the future.     

Detection of Anorectal and Cervicovaginal Chlamydia Trachomatis Infections following Azithromycin Treatment: Prospective Cohort Study with Multiple Time-Sequential Measures of rRNA,DNA, Quantitative Load and Symptoms

Background

Determination of Chlamydia trachomatis (Ct) treatment success is hampered by current assessment methods, which involve a single post-treatment measurement only. Therefore, we evaluated Ct detection by applying multiple laboratory measures on time-sequential post-treatment samples.

Methods

A prospective cohort study was established with azithromycin-treated (1000 mg) Ct patients (44 cervicovaginal and 15 anorectal cases). Each patient provided 18 self-taken samples pre-treatment and for 8 weeks post-treatment (response: 96%; 1,016 samples). Samples were tested for 16S rRNA (TMA), bacterial load (quantitative PCR; Chlamydia plasmid DNA) and type (serovar and multilocus sequence typing). Covariates (including behavior, pre-treatment load, anatomic site, symptoms, age, and menstruation) were tested for their potential association with positivity and load at 3–8 weeks using regression analyses controlling for repeated measures.

Findings

By day 9, Ct positivity decreased to 20% and the median load to 0.3 inclusion-forming units (IFU) per ml (pre-treatment: 170 IFU/ml). Of the 35 cases who reported no sex, sex with a treated partner or safe sex with a new partner, 40% had detection, i.e. one or more positive samples from 3–8 weeks (same Ct type over time), indicating possible antimicrobial treatment failure. Cases showed intermittent positive detection and the number of positive samples was higher in anorectal cases than in cervicovaginal cases. The highest observed bacterial load between 3–8 weeks post-treatment was 313 IFU/ml, yet the majority (65%) of positive samples showed a load of ≤2 IFU/ml. Pre-treatment load was found to be associated with later load in anorectal cases.

Conclusions

A single test at 3–8 weeks post-treatment frequently misses Ct. Detection reveals intermittent low loads, with an unknown risk of later complications or transmission. These findings warrant critical re-evaluation of the clinical management of single dose azithromycin-treated Ct patients and fuel the debate on defining treatment failure. Clinicaltrials.gov Identifier: NCT01448876.