IL-1R-associated kinase (IRAK)-1 is a critical mediator of TLR/IL-1R-induced activation of the transcription factor NF-kappaB. We previously described that a commonly occurring IRAK-1 variant haplotype, containing amino acid changes from serine to phenylalanine at position 196 and from leucine to serine at position 532, is associated with increased activation of NF-kappaB in LPS-stimulated neutrophils from patients with sepsis-induced acute lung injury and also higher mortality and more severe clinical outcomes in such patients. To investigate the underlying molecular mechanisms, we examined the ability of wild-type and variant IRAK-1 to modulate NF-kappaB activation. We found increased NF-kappaB transcriptional activity and expression of NF-kappaB-dependent proinflammatory cytokines in IL-1beta-stimulated IRAK-1-deficient cells transfected with variant IRAK-1 as compared with IRAK-1 wild type. IkappaB-alpha degradation was faster and p65 phosphorylation more prolonged after IL-1beta stimulation in cells expressing the IRAK-1 variant. However, IL-1-induced activation of MAPKs and nuclear translocation of NF-kappaB are comparable in both IRAK-1 variant- and IRAK-1 wild-type-expressing cells. Autophosphorylation of the IRAK-1 variant is greater than that found with wild-type IRAK-1. Additionally, variant IRAK-1 has greater interaction with TNFR-associated factor 6 than does wild-type IRAK-1. The enhanced activity of variant IRAK-1 appeared to be due to the alteration at aa 532, with only minimal effects being associated with change at aa 196. These results demonstrate that variant IRAK-1 is associated with alterations in multiple intracellular events that are likely to contribute to increased NF-kappaB activation and inflammatory responses in individuals with this IRAK-1 haplotype.
Uterine leiomyoma is a mesenchymal tumor composed of smooth muscle cells with fibrous tissues and many mast cells. Tranilast is known to suppress fibrosis or to work as a mast cell stabilizer and is reported to inhibit proliferation of vascular smooth muscle cells. In this study, we examined the effects of tranilast on cultured human leiomyoma cells in vitro to evaluate whether this agent has the potential to inhibit the growth of uterine leiomyomas. Tranilast inhibited the proliferation of cultured leiomyoma cells in a dose-dependent manner without any cytotoxic effect or induction of apoptosis. In association with the inhibitory effect, tranilast induced the cyclin-dependent kinase (CDK) inhibitor p21waf1 and tumor suppressor gene p53 and decreased CDK2 activity. These results suggest that tranilast arrests the proliferation of uterine leiomyoma cells at the G0/G1 phase, through the suppression of CDK2 activity via an induction of p21waf1 and p53. Tranilast was concluded to be a potent agent to inhibit proliferative activity of uterine leiomyoma cells.
Breast cancer is a leading cause of death in the United States. Despite some advances in treatment of primary breast cancer, patients with metastatic disease still have no chance of cure and conventional treatment modalities remain palliative. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced or metastatic breast cancer. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new approach for different tumor types. A critical factor determining the utility of Ad vectors for cancer gene therapy is the selectivity of their transgene expression in cancer cells. Strategies to restrict adenoviral-mediated transgene expression are important to avoid side effects due to gene transfer into healthy cells, especially the liver. However, currently available TSPs used in the context of Ad-based breast cancer targeting, are generally not tumor-specific but tissue specific. In this context, Heparanase (HPR), a heparan sulfate-specific endo-β-D-glucuronidase, is highly overexpressed in human cancers including breast cancer and has been shown to play an important role in tumor metastasis. In contrast, HPR expression is low or undetectable in differentiated, healthy tissue. To evaluate the utility of HPR as a TSP for breast cancer gene therapy, RT-PCR was performed to evaluate the expression of the HPR gene in various established breast cancer cell lines, primary human breast cancer tissue samples and normal breast cell lines. We constructed an Ad vector, AdHPR.luc, encoding luciferase under the control of the HPR promoter to determine relative activity in a variety of breast cancer, normal human breast cell lines and purified breast cancer tissue samples. An Ad vector containing the ubiquitously expressed CMV promoter (AdCMV.luc) was used as control. Biodistribution and liver tropism was evaluated after i.v. virus injection into a mouse model. Quantitative RT-PCR reveals a 4.5 – 44.6 fold, (p<0.05) increased expression of the HPR gene in several breast cancer cell lines compared to a normal breast control cell line. In primary breast cancer patient samples the average HPR mRNA copy number was significantly increased (90 fold, p<0.05). When compared to the ubiquitous CMV promoter, the HPR promoter showed a high level of activity in four breast cancer cell lines (5.5% – 12.7% compared to CMV) and primary breast cancer patient samples (8.8%– 14.4%), whereas activity in normal breast cells (1% compared to CMV) was low. The mouse liver toxicity and biodistribution profile of AdHPR.luc is significantly (2.3 fold, p<0.05) repressed compared to AdCMV.luc. In conclusion, we have identified the HPR promoter as a candidate for transcriptional targeting of breast cancer. In an Ad backbone, this promoter appears to be highly active in human breast cancer cells. These findings place the HPR pathway as a target for the development of breast cancer directed gene therapy strategies.
Obesity is a known risk factor for postmenopausal breast cancer and is associated with poorer prognosis for premenopausal and postmenopausal patients; however, the aetiological mechanisms are unknown. Preclinical studies support weight loss via caloric restriction and increased physical activity as a possible cancer control strategy, though few clinical studies have been conducted. We undertook a feasibility trial among women recently diagnosed with stage 0-II breast cancer hypothesising that presurgical weight loss would be feasible, safe and result in favourable changes in tumour markers and circulating biomarkers.A two-arm randomised controlled trial among 40 overweight or obese women, newly diagnosed with stage 0-II breast cancer and scheduled for surgery was planned. The attention control arm received upper body progressive resistance training and diet counselling to correct deficiencies in nutrient intake; the experimental arm received the same plus counselling on caloric restriction and aerobic exercise to achieve a weight loss of 0.68-0.919 kg/week. In addition to achieving feasibility benchmarks (accruing and retaining at least 80% of participants, and observing no serious adverse effects attributable to the intervention), we will explore the potential impact of an acute state of negative energy balance on tumour proliferation rates (Ki-67), as well as other tumour markers, serum biomarkers, gene expression, microbiome profiles and other clinical outcomes (eg, quality of life). Outcomes for the 2 study arms are compared using mixed models repeated-measures analyses.Ethics approval was received from the University of Alabama at Birmingham Institutional Review Board (Protocol number F130325009). Study findings will be disseminated through peer-reviewed publications. Given that this is one of the first studies to investigate the impact of negative energy balance directly on tumour biology in humans, larger trials will be pursued if results are favourable.NCT02224807; Pre-results.
