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Transient Receptor Potential Vanilloid 1 (TRPV1) activation induces autophagy in thymocytes through ROS-regulated AMPK and Atg4C pathways.
Get PDFAutophagy is a highly conserved process involved in lymphocyte development and differentiation. Herein, we demonstrated for the first time that triggering of Transient Receptor Potential Vanilloid 1 (TRPV1) by the specific agonist capsaicin (CPS) induces autophagy in mouse thymocytes. TRPV1-dependent autophagy required calcium influx and ROS generation resulting in AMP-activated kinase (AMPK) activation. CPS specifically increased autophagy related 4C (Atg4C) mRNA expression and induced oxidation of Atg4C protein by ROS generation. TRPV1-triggered autophagy was Atg6/Beclin-1-dependent, as demonstrated by the use of Beclin-1+/- transgenic mice, and involved ROS- and AMPK-mediated up-regulation of Beclin-1 expression. Autophagy is activated as pro-survival process since its inhibition triggered apoptosis of thymocytes: this effect was accompanied by down-regulation of Atg4C, Bcl-XL and immunity-related GTPase family M (Irgm1) mRNA expression, decreased Bcl-XL and Beclin-1 protein levels and caspase-3 activation, suggesting the existence of a molecular interplay between autophagic and apoptotic programs.
TRPV1 activation by CPS altered the expression of CD4 and CD8α antigens, inducing the development of a double positive subpopulation expressing lower levels of both receptors (DPdull), representing an intermediate stage of thymocyte maturation.
Interestingly, we found that DPdull represent the thymocyte subpopulation undergoing autophagy upon CPS treatment and that, when autophagy is inhibited, it becomes apoptotic. Our findings suggest that DPdull cells are able to respond either to survival or death signals and that TRPV1 channel dependent autophagy/apoptosis could play a major role in thymocyte development
Intracellular Calcium Levels and Cell Fate in Cancer: Interplay Between Senescence, Autophagy and Apoptosis
No full textEpithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women worldwide, with an overall 5 year survival rate below 30%. The low survival rate is associated with the persistence of cancer stem cells (CSCs) after chemotherapy. Therefore, CSC-targeting strategies are required for successful EOC treatment. Pan-human epidermal growth factor receptor 4 (HER4) and L-type calcium channels are highly expressed in ovarian CSCs, and treatment with the pan-HER inhibitor poziotinib or calcium channel blockers (CCBs) selectively inhibits the growth of ovarian CSCs via distinct molecular mechanisms. In this study, we tested the hypothesis that combination treatment with poziotinib and CCBs can synergistically inhibit the growth of ovarian CSCs. Combined treatment with poziotinib and manidipine (an L-type CCB) synergistically suppressed ovarian CSC sphere formation and viability compared with either drug alone. Moreover, combination treatment synergistically reduced the expression of stemness markers, including CD133, KLF4, and NANOG, and stemness-related signaling molecules, such as phospho-STAT5, phospho-AKT, phospho-ERK, and Wnt/β-catenin. Moreover, poziotinib with manidipine dramatically induced apoptosis in ovarian CSCs. Our results suggest that the combinatorial use of poziotinib with a CCB can effectively inhibit ovarian CSC survival and functionVarious derivatives that mimic ceramide structures by introducing a triazole to connect the aminodiol moiety and long alkyl chain have been synthesized and screened for their anti-leukemia activity. SPS8 stood out among the derivatives, showing cytotoxic selectivity between leukemic cell lines and human peripheral blood mononuclear cells (about ten times). DAPI nuclear staining and H&E staining revealed DNA fragmentation under the action of SPS8. SPS8 induced an increase in intracellular Ca2+ levels and mitochondrial stress in HL-60 cells identified by the loss of mitochondrial membrane potential, transmission electron microscopy (TEM) examination, and altered expressions of Bcl-2 family proteins. SPS8 also induced autophagy through the detection of Atg5, beclin-1, and LC3 II protein expression, as well as TEM examination. Chloroquine, an autophagy inhibitor, promoted SPS8-induced apoptosis, suggesting the cytoprotective role of autophagy in hindering SPS8 from apoptosis. Furthermore, SPS8 was shown to alter the expressions of a variety of genes using a microarray analysis and volcano plot filtering. A further cellular signaling pathways analysis suggested that SPS8 induced several cellular processes in HL-60, including the sterol biosynthesis process and cholesterol biosynthesis process, and inhibited some cellular pathways, in which STAT3 was the most critical nuclear factor. Further identification revealed that SPS8 inhibited the phosphorylation of STAT3, representing the loss of cytoprotective activity. In conclusion, the data suggest that SPS8 induces both apoptosis and autophagy in leukemic cells, in which autophagy plays a cytoprotective role in impeding apoptosis. Moreover, the inhibition of STAT3 phosphorylation may support SPS8-induced anti-leukemic activity.Calcium (Ca2+) is a major second messenger in cells and is essential for the fate and survival of all higher organisms. Different Ca2+ channels, pumps, or exchangers regulate variations in the duration and levels of intracellular Ca2+, which may be transient or sustained. These changes are then decoded by an elaborate toolkit of Ca2+-sensors, which translate Ca2+ signal to intracellular operational cell machinery, thereby regulating numerous Ca2+-dependent physiological processes. Alterations to Ca2+ homoeostasis and signaling are often deleterious and are associated with certain pathological states, including cancer. Altered Ca2+ transmission has been implicated in a variety of processes fundamental for the uncontrolled proliferation and invasiveness of tumor cells and other processes important for cancer progression, such as the development of resistance to cancer therapies. Here, we review what is known about Ca2+ signaling and how this fundamental second messenger regulates life and death decisions in the context of cancer, with particular attention directed to cell proliferation, apoptosis, and autophagy. We also explore the intersections of Ca2+ and the therapeutic targeting of cancer cells, summarizing the therapeutic opportunities for Ca2+ signal modulators to improve the effectiveness of current anticancer therapies.Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors
TRPV channels in tumor growth and progression
No full textTransient receptor potential (TRP) channels affect several physiological and pathological processes. In particular, TRP channels have been recently involved in the triggering of enhanced proliferation, aberrant differentiation, and resistance to apoptotic cell death leading to the uncontrolled tumor invasion. About thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like). Among these channel families, the TRPC, TRPM, and TRPV families have been mainly correlated with malignant growth and progression. The aim of this review is to summarize data reported so far on the expression and the functional role of TRPV channels during cancer growth and progression. TRPV channels have been found to regulate cancer cell proliferation, apoptosis, angiogenesis, migration and invasion during tumor progression, and depending on the stage of the cancer, up- and down-regulation of TRPV mRNA and protein expression have been reported. These changes may have cancer promoting effects by increasing the expression of constitutively active TRPV channels in the plasma membrane of cancer cells by enhancing Ca(2+)-dependent proliferative response; in addition, an altered expression of TRPV channels may also offer a survival advantage, such as resistance of cancer cells to apoptotic-induced cell death. However, recently, a role of TRPV gene mutations in cancer development, and a relationship between the expression of specific TRPV gene single nucleotide polymorphisms and increased cancer risk have been reported. We are only at the beginning, a more deep studies on the physiopathology role of TRPV channels are required to understand the functional activity of these channels in cancer, to assess which TRPV proteins are associated with the development and progression of cancer and to develop further knowledge of TRPV proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer
Role of Capsaicin in Oxidative Stress and Cancer
No full textCapsaicin has been shown to induces apoptosis in various trasformed cell types in vitro and in vivo
Capsaicin promotes a more aggressive gene expression phenotype and invasiveness in null-TRPV1 urothelial cancer cells.
Get PDFCapsaicin (CPS) has been found to exhibit either tumor promoting or suppressing effects, many of which are mediated by the specific transient receptor potential vanilloid type-1 (TRPV1). Herein, we provide evidence that CPS treatment induced a more aggressive gene phenotype and invasiveness in 5637 cells-lacking TRPV1 receptor. CPS treatment of 5637 cells induced upregulation of pro-angiogenetic (angiopoietin 1, angiopoietin 2 and vascular endothelial growth factor), pro-invasive and pro-metastatic genes (MMP1, MMP9, TIMP1, TIMP3, granzyme A (GZMA), NM23A and S100A) with a downregulation of apoptotic genes (Fas/CD95 and tumor necrosis factor receptor superfamily member 1A). CPS increased the invasiveness of 5637 cells by triggering IGF (insulin-like growth factor)-1 release, GZMA and MMP9 activation, α-tubulin disassembly and cytoskeleton degradation. Finally, in order to evaluate the relationship between the lack of TRPV1 expression and increased CPS-induced invasiveness, we transfected 5637 cells with the TRPV1 complementary DNA (cDNA) sequence. We found that TRPV1-expressing cells show CPS-mediated calcium level increase, growth inhibition and apoptosis. Moreover, CPS-induced migration and MMP9 activation were reverted, suggesting an inhibitory role played by TRPV1 in urothelial cancer cell invasion and metastasis
Normal human urothelial cell lines express onabotulinumtoxinA SV2 high affinity receptors.
No full textINTRODUCTION & OBJECTIVES: Botulinum A toxin (BoNT/A) internalization within the target cells requires the binding with high affinity receptors (Synaptic Vesicle Proteins type 2 -SV2). To date, 3 different isoforms of SV2 have been identified in synaptic and
endocrine tissues. Bladder SV2 immunoreactivity has been previously identified in parasympathetic, sympathetic and sensory fibers, but not
into bladder urothelial cells. The aim of this study was to evaluate the expression of SV2 in human non neoplastic urothelial cells at genic
and protein levels.
MATERIAL & METHODS: The study was performed in normal human urothelial cell lines (NHUCs). Expression of the 3 SV2 isoforms (SV2A,
-B –C) was investigated by quantitative Real Time PCR (qPCR) and Western blot analysis. In order to identify SV2 subcellular localization,
NHUCs were fractionated and proteins from different cellular compartments including membrane, cytoplasm, soluble nuclear and
cytoskeleton were separated by SDS-PAGE and probed with anti SV2-A, -B and -C Abs. Mouse brain tissue was used as positive control and
ß actin was used as loading control.
RESULTS: qPCR demonstrated the presence of SV2-A, -B and -C mRNAs in NHUCs. Higher levels of SV2C (about 1.8 folds) were
expressed as compared to SV2A isoform mRNA levels. In addition, SV2B exhibited a 20- and 30-fold reduction in the amount of mRNA
levels in comparison with SV2A and SV2C, respectively (Fig. 1). Western blot analysis showed bands with proper molecular weights,
likely corresponding to SV2-A, -B and –C, in lysates from NHUCs and mouse brain tissue. All 3 isoforms were found to be accumulated
primarily in the cytoplasmic extract, weakly expressed in the membrane fraction and were not detected in soluble nuclear and
cytoskeletal compartments.
CONCLUSIONS: We demonstrated for the first time the presence of SV2 BoNT/A high affinity receptors on the plasmatic membranes of
NHUCs. Recent observations suggested a crucial role for the urothelium in modulating underlying sensory fibers. This may be due
not only to the urothelial release of Ach, but also of other neurotransmitters, as ATP, CGRP and Substance P. BoNT/A could act
through SV2-mediated internalization on human urothelial cells thus blocking neurotransmitters' exocytosis and controlling afferent
transmission and pain. The higher level of SV2C, as compared to the other isoforms, is consistent with the previously described most robust BoNT/A binding activity with SV2C
Present and Future of Tyrosine Kinase Inhibitors in Renal Cell Carcinoma: Analysis of Hematologic Toxicity.
No full textTyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of renal cell carcinoma (RCC) patients. The use of these agents requires early and appropriate management of side effects such as hematologic adverse events (HAE), in order to avoid unnecessary dose reductions and transitory or definitive treatment discontinuations. Beyond the increased infective risk, myelosuppression contributes to TKI-related fatigue, thus reducing both patients' quality of life and overall survival (OS). However, the frequency and severity of myelosuppression vary among sunitinib, sorafenib, pazopanib and axitinib, based on their different kinase selectivity. Their activity against fms-related tyrosine kinase 3 (FLT3 or CD135) and c-kit, which are essential for survival and differentiation of hemopoietic progenitor cells, is critical to determine the hematologic toxicity profiles. This review describes the molecular mechanisms underlying the TKI effects exerted on hematopoiesis and immune response and related recent patents, of drugs already approved or still under evaluation in RCC, highlighting the potential impact of these effects on tumor response to treatment
Assessment of Botulinum a Toxin High Affinity SV2 Receptors on Normal Human Urothelial Cells.
Get PDFThe clinical use of Botulinum A toxin (BoNT/A) is based on its ability to block the vesicular release of Acetilcholine (Ach) and other neurotransmitters at the level of the neuronal plasmatic membrane. This process requires the internalization of the neurotoxin within the target cell, what it happens by means of the binding with high affinity receptors. These receptors are the synaptic vesicle proteins type 2 (SV2). To date three different types of SV2 receptors have been identified in synaptic structures and endocrine tissues
Epigenetic, Genetic, and Acquired Regulation of Cav3 T-Type Calcium Channel Expression and Function in Tumor Growth and Progression
Get PDFT-type Ca2+ channels are a specific channel family controlling proliferation, differentiation, angiogenesis,
and invasion of tumor cells. Molecular biology has identified three main subfamilies of α1 subunits, called
Cav1, Cav2, and Cav3. The third subfamily contains three members, called T-types: Cav3.1 (α1G), Cav3.2
(α1H), and Cav3.3 (α1I). The Cav3 channels are expressed in normal tissues throughout the body as well as
in different types of tumors such as breast, glioma/neuroblastomas, colorectal, gastric, hepatic and prostate
tumors, T cell leukemia, retinoblastoma (RB), and phaeochromocytoma. It has been shown that increased
functional expression of Cav3 channels plays a role in abnormal proliferation of tumor cells. In addition, a
crosstalk between the Rho-ROCK pathway and Cav3 channels in tumor cell migration and invasion has
been demonstrated. Cav3 expression is strictly regulated during cell differentiation and tumor formation.
Inactivation of Cav3 genes by aberrant methylation plays a major role in tumor development and
progression. In this regard, since hypermethylation of CpG islands is thought to be the major epigenetic
modification repressing gene transcription, Cav3.1 has been regarded as a candidate tumor suppressor gene.
In contrast, CpG sites in the Cav3.2 gene were hypomethylated, and thus this gene is considered to be a
candidate oncogene, regulated by methylation. Finally, Cav3 functional diversity may also result from the
generation of splice variants. Alterations in the expression of different splice variants, and their expression
during tumor development and progression, may trigger variety in Ca2+ signaling, which may contribute to
the generation of more aggressive tumor clones
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