Chemosensitivity of radioresistant cells in the multicellular spheroids of A549 lung adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>The relapse of cancer after radiotherapy is a clinical knotty problem. Previous studies have demonstrated that the elevation of several factors is likely in some way to lead to the development of treatment tolerance, so it is necessary to further explore the problem of re-proliferated radioresistant cells to chemotherapeutic agents. In the present study, we aimed to investigate the chemosensitivity of radioresistant cells originated from the multicellular spheroids of A549 lung adenocarcinoma.</p> <p>Methods</p> <p>After irradiated with 25 Gy of 6 MV X-ray to A549 multicellular spheroids, whose 10th re-proliferated generations were employed as radioresistant cells, and the control groups were A549 parental cells and MCF7/VCR resistant cells. The chemo-sensitivity test was made by six kinds of chemotherapeutic drugs which were DDP, VDS, 5-Fu, HCP, MMC and ADM respectively, while verapamil (VPL) was used as the reversal agent. Then the treatment effect was evaluated by MTT assay, and the multidrug resistant gene expressions of <it>mdr1 </it>and <it>MRP </it>were measured by RT-PCR.</p> <p>Results</p> <p>Both A549 parental cells and A549 derived radioresistant cells were resistant to DDP, but sensitive to VDS, 5-Fu, HCP, MMC and ADM. The inhibitory rates of VPL to these two types of cell were 98% and 25% respectively (P < 0.001). In addition, without drugs added, the absorbance value (A value) of A549 parental cells was 2-folds higher than that of their radioresistant cells (P < 0.001). As to the MCF7/VCR cells, they were resistant to DDP and VDS, but slight sensitive to MMC, ADM, 5-Fu, and HCP with 80% of inhibitory rate to VPL. The subsequent RT-PCR demonstrated that the <it>Mdr1</it>/β2-MG and <it>MRP</it>/β2-MG of all A549 cells were about 0 and 0.7 respectively, and those of MCF7/VCR cells were 35 and 4.36.</p> <p>Conclusion</p> <p>The chemosensitivity of A549 radioresistant cells had not changed markedly, and the decreased sensitivity to VPL could not be explained by the gene expression of <it>mdr1 </it>and <it>MRP</it>. It is possible that the changes in the cell membrane and decreased proliferate ability might be attributed to the resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to radioresistant cells. Therefore, the new biological strategy needs to be developed to treat recurring radioresistant tumor in combination with chemotherapy.</p

Wanted dead or alive : high diversity of macroinvertebrates associated with living and ’dead’ Posidonia oceanica matte

The Mediterranean endemic seagrass Posidonia oceanica forms beds characterised by a dense leaf canopy and a thick root-rhizome ‘matte’. Death of P. oceanica shoots leads to exposure of the underlying matte, which can persist for many years, and is termed ‘dead’ matte. Traditionally, dead matte has been regarded as a degraded habitat. To test whether this assumption was true, the motile macroinvertebrates of adjacent living (with shoots) and dead (without shoots) matte of P. oceanica were sampled in four different plots located at the same depth (5–6 m) in Mellieha Bay, Malta (central Mediterranean). The total number of species and abundance were significantly higher (ANOVA; P<0.05 and P<0.01, respectively) in the dead matte than in living P. oceanica matte, despite the presence of the foliar canopy in the latter. Multivariate analysis (MDS) clearly showed two main groups of assemblages, corresponding to the two matte types. The amphipods Leptocheirus guttatus and Maera grossimana, and the polychaete Nereis rava contributed most to the dissimilarity between the two different matte types. Several unique properties of the dead matte contributing to the unexpected higher number of species and abundance of motile macroinvertebrates associated with this habitat are discussed. The findings have important implications for the conservation of bare P. oceanica matte, which has been generally viewed as a habitat of low ecological value.peer-reviewe

Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation

Background: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine serum

Interferon-α Abrogates Tolerance Induction by Human Tolerogenic Dendritic Cells

BACKGROUND: Administration of interferon-α (IFN-α) represents an approved adjuvant therapy as reported for malignancies like melanoma and several viral infections. In malignant diseases, tolerance processes are critically involved in tumor progression. In this study, the effect of IFN-α on tolerance induction by human tolerogenic dendritic cells (DC) was analyzed. We focussed on tolerogenic IL-10-modulated DC (IL-10 DC) that are known to induce anergic regulatory T cells (iTregs). METHODOLOGY/PRINCIPAL FINDINGS: IFN-α promoted an enhanced maturation of IL-10 DC as demonstrated by upregulation of the differentiation marker CD83 as well as costimulatory molecules. IFN-α treatment resulted in an increased capacity of DC to stimulate T cell activation compared to control tolerogenic DC. We observed a strengthened T cell proliferation and increased IFN-γ production of CD4(+) and CD8(+) T cells stimulated by IFN-α-DC, demonstrating a restoration of the immunogenic capacity of tolerogenic DC in the presence of IFN-α. Notably, restimulation experiments revealed that IFN-α treatment of tolerogenic DC abolished the induction of T cell anergy and suppressor function of iTregs. In contrast, IFN-α neither affected the priming of iTregs nor converted iTregs into effector T cells. CONCLUSIONS/SIGNIFICANCE: IFN-α inhibits the induction of T cell tolerance by reversing the tolerogenic function of human DC

Evolution of bisphosphonate-related osteonecrosis of the jaw in patients with multiple myeloma and Waldenstrom's macroglobulinemia: a retrospective multicentric study

Bisphosphonates (BPs) are used intravenously to treat cancer-related conditions for the prevention of pathological fractures. Osteonecrosis of the jaw (BRONJ) is a rare complication reported in 4–15% of patients. We studied, retrospectively, 55 patients with multiple myeloma or Waldenstrom's macroglobulinemia followed up from different haematological departments who developed BRONJ. All patients were treated with BPs for bone lesions and/or fractures. The most common trigger for BRONJ was dental alveolar surgery. After a median observation of 26 months, no death caused by BRONJ complication was reported. In all, 51 patients were treated with antibiotic therapy, and in 6 patients, this was performed in association with surgical debridement of necrotic bone, in 16 with hyperbaric O2 therapy/ozonotherapy and curettage and in 12 with sequestrectomy and O2/hyperbaric therapy. Complete response was observed in 20 cases, partial response in 21, unchanged in 9 and worsening in 3. The association of surgical treatment with antibiotic therapy seems to be more effective in eradicating the necrotic bone than antibiotic treatment alone. O2 hyperbaric/ozonotherapy is a very effective treatment. The cumulative dosage of BPs is important for the evolution of BRONJ. Because the most common trigger for BRONJ was dental extractions, all patients, before BP treatment, must achieve an optimal periodontal health

Similar TKA designs with differences in clinical outcome: A randomized, controlled trial of 77 knees with a mean follow-up of 6 years

Contains fulltext : 96347.pdf (publisher's version ) (Open Access)Background and purpose To try to improve the outcome of our TKAs, we started to use the CKS prosthesis. However, in a retrospective analysis this design tended to give worse results. We therefore conducted a randomized, controlled trial comparing this CKS prosthesis and our standard PFC prosthesis. Because many randomized studies between different TKA concepts generally fail to show superiority of a particular design, we hypothesized that these seemingly similar designs would not lead to any difference in clinical outcome. Patients and methods 82 patients (90 knees) were randomly allocated to one or other prosthesis, and 39 CKS prostheses and 38 PFC prostheses could be followed for mean 5.6 years. No patients were lost to follow-up. At each follow-up, patients were evaluated clinically and radiographically, and the KSS, WOMAC, VAS patient satisfaction scores and VAS for pain were recorded. Results With total Knee Society score (KSS) as primary endpoint, there was a difference in favor of the PFC group at final follow-up (p = 0.04). Whereas there was one revision in the PFC group, there were 6 revisions in the CKS group (p = 0.1). The survival analysis with any reoperation as endpoint showed better survival in the PFC group (97% (95% CI: 92-100) for the PFC group vs. 79% (95% CI: 66-92) for the CKS group) (p = 0.02). Interpretation Our hypothesis that there would be no difference in clinical outcome was rejected in this study. The PFC system showed excellent results that were comparable to those in previous reports. The CKS design had differences that had considerable negative consequences clinically. The relatively poor results have discouraged us from using this design

PI3K/AKT is involved in mediating survival signals that rescue Ewing tumour cells from fibroblast growth factor 2-induced cell death

While in vitro studies had shown that fibroblast growth factor 2 (FGF2) can induce cell death in Ewing tumours, it remained unclear how Ewing tumour cells survive in vivo within a FGF2-rich microenvironment. Serum- and integrin-mediated survival signals were, therefore, studied in adherent monolayer and anchorage-independent colony cell cultures. In a panel of Ewing tumour cell lines, either adhesion to collagen or exposure to serum alone only had a minor protective effect against FGF2. However, both combined led to complete resistance to 5 ng ml−1 FGF2 in three of four FGF2-sensitive cell lines (RD-ES, RM-82 and WE-68), and to an increased survival as compared to other culture conditions in TC-71 cells. Inhibition studies with LY294002 demonstrated that the serum signal is mediated via the phosphoinositide 3-OH kinase/AKT pathway. Thus, Ewing tumour cells escape FGF2-induced cell death by modulating FGF2 signalling. The tumour microenvironment provides the necessary survival signals by integrin-mediated adhesion and soluble serum factor(s). These survival signals warrant further investigation as a potential resistance mechanism to other apoptosis-inducing agents in vivo

Bisphosphonates as antimyeloma drugs

In patients with symptomatic multiple myeloma (MM), bisphosphonate (BP) treatment has been widely used to prevent bone loss and preserve skeletal health because of its proven effects on inhibiting osteoclast-mediated bone resorption. In addition to their effects on osteoclasts, it is becoming increasingly evident that BPs may have additional effects on the bone microenvironment and cells other than osteoclasts that may potentially inhibit the development and progression of MM. This review focuses on the pathophysiology of MM with an emphasis on the events that drive MM progression within the bone and the mechanisms by which BPs may inhibit specific processes. The underlying molecular mechanisms that drive the modulation of cellular fate and function and consequent physiological outcomes are described. Direct effects on myeloma cell growth and survival and the interactions between myeloma cells and the bone microenvironment are discussed. Clinical evidence of the antimyeloma effects of BPs is emerging and is also reviewed

H4 Histamine Receptors Mediate Cell Cycle Arrest in Growth Factor-Induced Murine and Human Hematopoietic Progenitor Cells

The most recently characterized H4 histamine receptor (H4R) is expressed preferentially in the bone marrow, raising the question of its role during hematopoiesis. Here we show that both murine and human progenitor cell populations express this receptor subtype on transcriptional and protein levels and respond to its agonists by reduced growth factor-induced cell cycle progression that leads to decreased myeloid, erythroid and lymphoid colony formation. H4R activation prevents the induction of cell cycle genes through a cAMP/PKA-dependent pathway that is not associated with apoptosis. It is mediated specifically through H4R signaling since gene silencing or treatment with selective antagonists restores normal cell cycle progression. The arrest of growth factor-induced G1/S transition protects murine and human progenitor cells from the toxicity of the cell cycle-dependent anticancer drug Ara-C in vitro and reduces aplasia in a murine model of chemotherapy. This first evidence for functional H4R expression in hematopoietic progenitors opens new therapeutic perspectives for alleviating hematotoxic side effects of antineoplastic drugs

Evidence for Epithelial-Mesenchymal Transition in Cancer Stem Cells of Head and Neck Squamous Cell Carcinoma

Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1+ cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1+ cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44+/CD24− was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%–33% with the CD44+/CD24−/ALDH1+ cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis