Lídia M. Andrade

Nuclear inositol 1,4,5-trisphosphate is a necessary and conserved signal for the induction of both pathological and physiological cardiomyocyte hypertrophy.

It is well established that inositol 1,4,5-trisphosphate (IP3) dependent Ca(2+) signaling plays a crucial role in cardiomyocyte hypertrophy. However, it is not yet known whether nuclear IP3 represents a Ca(2+) mobilizing pathway involved in this process. The goal of the current work was to investigate the specific role of nuclear IP3 in cardiomyocyte hypertrophic response. In this work, we used an adenovirus construct that selectively buffers IP3 in the nuclear region of neonatal cardiomyocytes. We showed for the first time that nuclear IP3 mediates endothelin-1 (ET-1) induced hypertrophy. We also found that both calcineurin (Cn)/nuclear factor of activated T Cells (NFAT) and histone deacetylase-5 (HDAC5) pathways require nuclear IP3 to mediate pathological cardiomyocyte growth. Additionally, we found that nuclear IP3 buffering inhibited insulin-like growth factor-1 (IGF-1) induced hypertrophy and prevented reexpression of fetal gene program. Together, these results demonstrated that nuclear IP3 is an essential and a conserved signal for both pathological and physiological forms of cardiomyocyte hypertrophy.

The insulin receptor translocates to the nucleus to regulate cell proliferation in liver

Insulins metabolic effects in the liver are widely appreciated, but insulins ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca2+ signals within the nucleus regulate cell proliferation, we investigated whether insulins mitogenic effects result from activation of Ca2+‐signaling pathways by IRs within the nucleus. Insulin‐induced increases in Ca2+ and cell proliferation depended upon clathrin‐ and caveolin‐dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,‐trisphosphate (InsP3) in the nucleus, whereas insulins metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus. Conclusion: These findings provide evidence that insulins mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3‐dependent Ca2+‐signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease. (Hepatology 2014;58:274–283)

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

Subcellular Ca2+ signals control a variety of responses in the liver. For example, mitochondrial Ca2+ (Ca  mit2+ ) regulates apoptosis, whereas Ca2+ in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca  mit2+ also affects liver regeneration. The Ca2+‐buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin–mitochondrial targeting sequence–green fluorescent protein (PV‐MITO‐GFP). This construct properly localized to and effectively buffered Ca2+ signals in the mitochondrial matrix. Additionally, the expression of PV‐MITO‐GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl‐2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2–associated X protein (bax), apoptotic peptidase activating factor 1, and caspase‐6]. PV‐MITO‐GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca  mit2+ buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl‐2 and the decreased expression of bax. Conclusion: Together, these results reveal an essential role for Ca  mit2+ in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis. (HEPATOLOGY 2011;)

Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity

BackgroundAdenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death.ResultsAPAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure.ConclusionWe suggest that under pathological conditions, ATP may act not only an immune system activator, but also as a paracrine direct cytotoxic DAMP through the dysregulation of Ca2+ homeostasis.

Nucleoplasmic Calcium Buffering Sensitizes Human Squamous Cell Carcinoma to Anticancer Therapy

Background: Calcium (Ca2+) signaling within the nucleus is known to play a crucial role in cell proliferation. The aim of this study was to investigate whether nuclear Ca2+ buffering could improve the antitumor effect of X-rays therapy on Human Squamous Cell Carcinoma (HSCC). Methods: For these purpose, we developed an experimental protocol that simulated clinical radiotherapy and prevented bystander effects of irradiation. HSCC, A431 cell line, was submitted to 10Gy cumulative X-rays therapy alone (XR Cd 10Gy) or in association with the strategy that selectively buffer nuclear Ca 2+ (Ca 2+ n) signaling. Results: Upon Ca 2+ n buffering, A431 cell proliferation rate decreased significantly as compared to control. Cell cycle analysis showed that association of Ca2+ n buffering with XR Cd 10Gy increased the percentage of A431 cells at G 2 /M and did not increase nuclear/mitochondrial DNA damages. Nonetheless, Ca 2+ n buffering prevented the increase of the radioresistance-related biomarker ADAM-17 expression and EGFR activation induced by irradiation. Furthermore, the association therapy almost completely abolished cell survival fraction even using approximately half of the X-rays cumulative dose Conclusions: Nuclear Ca 2+ buffering sensitizes human squamous cell carcinoma to X - rays irradiation treatment.

Decoding Calcium Signaling Across the Nucleus

Calcium (Ca(2+)) is an important multifaceted second messenger that regulates a wide range of cellular events. A Ca(2+)-signaling toolkit has been shown to exist in the nucleus and to be capable of generating and modulating nucleoplasmic Ca(2+) transients. Within the nucleus, Ca(2+) controls cellular events that are different from those modulated by cytosolic Ca(2+). This review focuses on nuclear Ca(2+) signals and their role in regulating physiological and pathological processes.

Radiation oncology in vitro: trends to improve radiotherapy through molecular targets.

Much has been investigated to improve the beneficial effects of radiotherapy especially in that case where radioresistant behavior is observed. Beyond simple identification of resistant phenotype the discovery and development of specific molecular targets have demonstrated therapeutic potential in cancer treatment including radiotherapy. Alterations on transduction signaling pathway related with MAPK cascade are the main axis in cancer cellular proliferation even as cell migration and invasiveness in irradiated tumor cell lines; then, for that reason, more studies are in course focusing on, among others, DNA damage enhancement, apoptosis stimulation, and growth factors receptor blockages, showing promising in vitro results highlighting molecular targets associated with ionizing radiation as a new radiotherapy strategy to improve clinical outcome. In this review we discuss some of the main molecular targets related with tumor cell proliferation and migration as well as their potential contributions to radiation oncology improvements.

HDR brachytherapy decreases proliferation rate and cellular progression of a radioresistant human squamous cell carcinoma in vitro

Abstract Purpose: To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters. Materials and methods: An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method. Results: Significant decreasing cellular proliferation rate (p < 0.05) as well as reduced survival fraction (p < 0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the G2/M phase (32.3 ± 1.5%) in the irradiated group as compared with untreated cells (8.22 ± 1.2%) was detected. Also, a significant (p < 0.0001) nuclei shrinking in irradiated cells without evidence of necrosis or apoptosis was found. Conclusion: HDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G2/M cell cycle arrest.

Synthesis, Purification and Functionalization of Carbon Nanotubes for Biotechnological Applications

This chapter addresses the initial steps one has to give to explore the unique properties of carbon nanotubes in biomedical applications, which are their synthesis, purification and functionalization. We start with a brief description of the structure and properties, and we summarize the synthesis methods. The major issues are then addressed, which are purification and functionalization for bioapplications. We review the different methods, highlighting their advantages and disadvantages.