Porphyrin-BODIPY Dyad: Enhancing Photodynamic Inactivation via Antenna Effect.

A porphyrin-BODIPY dyad (P-BDP) was obtained through covalent bonding, featuring a two-segment design comprising a light-harvesting antenna system connected to an energy acceptor unit. The absorption spectrum of P-BDP resulted from an overlap of the individual spectra of its constituent parts, with the fluorescence emission of the BODIPY unit experiencing significant quenching (96 %) due to the presence of the porphyrin unit. Spectroscopic, computational, and redox investigations revealed a competition between photoinduced energy and electron transfer processes. The dyad demonstrated the capability to sensitize both singlet molecular oxygen and superoxide radical anions. Additionally, P-BDP effectively induced the photooxidation of L-tryptophan. In suspensions of Staphylococcus aureus cells, the dyad led to a reduction of over 3.5 log (99.99 %) in cell survival following 30 min of irradiation with green light. Photodynamic inactivation caused by P-BDP was also extended to the individual bacterium level, focusing on bacterial cells adhered to a surface. This dyad successfully achieved the total elimination of the bacteria upon 20 min of irradiation. Therefore, P-BDP presents an interesting photosensitizing structure that takes advantage of the light-harvesting antenna properties of the BODIPY unit combined with porphyrin, offering potential to enhance photoinactivation of bacteria.

A Potential Boron Neutron Capture Therapy Agent Selectively Suppresses High-Grade Glioma: In Vitro and in Vivo Exploration.

Glioblastoma (GBM), as the most central nervous system (CNS) intractable disease, has spoiled millions of lives due to its high mortality. Even though several efforts have been made, the existing treatments have had limited success. In this sense, we studied a lead compound, the boron-rich selective epidermal growth factor receptor (EGFR)-inhibitor hybrid 1, as a potential drug for GBM treatment. For this end, we analyzed the in vitro activity of hybrid 1 in a glioma/primary astrocytes coculture, studying cellular death types triggered by treatment with this compound and its cellular localizations. Additionally, hybrid 1 concentrated boron in glioma cells selectively and more effectively than the boron neutron capture therapy (BNCT)-clinical agent 10B-l-boronophenylalanine and thus displayed a better in vitro-BNCT effect. This encouraged us to analyze hybrid 1 in vivo. Therefore, immunosuppressed mice bearing U87 MG human GBM were treated with both 1 and 1 encapsulated in a modified liposome (recognized by brain-blood barrier peptide transporters), and we observed a potent in vivo per se antitumor activity (tumor size decrease and animal survival increase). These data demonstrate that 1 could be a promising new targeted therapy for GBM.

Boron-Containing Compounds for Prevention, Diagnosis, and Treatment of Human Metabolic Disorders.

The application of natural and synthetic boron-containing compounds (BCC) in biomedical field is expanding. BCC have effects in the metabolism of living organisms. Some boron-enriched supplements are marketed as they exert effects in the bone and skeletal muscle; but also, BCC are being reported as acting on the enzymes and transporters of membrane suggesting they could modify the carbohydrate metabolism linked to some pathologies of high global burden, as an example is diabetes mellitus. Also, some recent findings are showing effects of BCC on lipid metabolism. In this review, information regarding the effects and interaction of these compounds was compiled, as well as the potential application for treating human metabolic disorders is suggested.

A store-operated Ca2+-entry in Trypanosoma equiperdum: Physiological evidences of its presence.

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca2+ entry are scarce. Even more, to date the presence of a store-operated Ca2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca2+-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca2+-entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum.

Self-Sterilizing 3D-Printed Polylactic Acid Surfaces Coated with a BODIPY Photosensitizer.

Herein, we report the use of polylactic acid coated with a halogenated BODIPY photosensitizer (PS) as a novel self-sterilizing, low-cost, and eco-friendly material activated with visible light. In this article, polymeric surfaces were 3D-printed and treated with the PS using three simple methodologies: spin coating, aerosolization, and brush dispersion. Our studies showed that the polymeric matrix remains unaffected upon addition of the PS, as observed by dynamic mechanical analysis, Fourier transform infrared, scanning electron microscopy (SEM), and fluorescence microscopy. Furthermore, the photophysical and photodynamic properties of the dye remained intact after being adsorbed on the polymer. This photoactive material can be reused and was successfully inactivating methicillin-resistant Staphylococcus aureus and Escherichia coli in planktonic media for at least three inactivation cycles after short-time light exposure. A real-time experiment using a fluorescence microscope showed how bacteria anchored to the antimicrobial surface were inactivated within 30 min using visible light and low energy. Moreover, the material effectively eradicated these two bacterial strains on the first stage of biofilm formation, as elucidated by SEM. Unlike other antimicrobial approaches that implement a dissolved PS or non-sustainable materials, we offer an accessible green and economic alternative to acquire self-sterilizing surfaces with any desired shape.

Effects of boron-containing compounds in the fungal kingdom.

BACKGROUND: The number of known boron-containing compounds (BCCs) is increasing due to their identification in nature and innovative synthesis procedures. Their effects on the fungal kingdom are interesting, and some of their mechanisms of action have recently been elucidated. METHODS: In this review, scientific reports from relevant chemistry and biomedical databases were collected and analyzed. RESULTS: It is notable that several BCC actions in fungi induce social and economic benefits for humans. In fact, boric acid was traditionally used for multiple purposes, but some novel synthetic BCCs are effective antifungal agents, particularly in their action against pathogen species, and some were recently approved for use in humans. Moreover, most reports testing BCCs in fungal species suggest a limiting effect of these compounds on some vital reactions. CONCLUSIONS: New BCCs have been synthesized and tested for innovative technological and biomedical emerging applications, and new interest is developing for discovering new strategic compounds that can act as environmental or wood protectors, as well as antimycotic agents that let us improve food acquisition and control some human infections.

Revealing ROS Production by Antibiotics and Photosensitizers in Biofilms: A Fluorescence Microscopy Approach.

Reactive oxygen species (ROS) production within biofilms is studied with a simple and easy setup based on fluorescence microscopy. Herein, a biofilm is exposed to different ROS inducers: a bactericidal antibiotic (ciprofloxacin) and a BODIPY-based photosensitizer (I2B-OAc). Real-time ROS induction in the core of the biofilms is monitored utilizing two fluorescent reporters-AMDA and H2DCFDA-the first one with selectivity toward singlet oxygen (1O2) and the latest for other ROS (O2•-, H2O2, and OH•-). A point-by-point methodology is reported, starting with the sample preparation all the way through the microscope setup and, finally, processing of the images.

Lipid Peroxidation Assay Using BODIPY-Phenylbutadiene Probes: A Methodological Overview.

The assessment of reactive oxygen species has increasing importance in biomedical sciences, due to their biological role in signaling pathways and induction of cell damage at low and high concentrations, respectively. Detection of lipid peroxidation with sensing probes such as some BODIPY dyes has now wide application in studies using fluorescent microplate readers, flow cytometry, and fluorescence microscopy. Two phenylbutadiene derivatives of BODIPY are commonly used as peroxidation probes, non-oxidized probes and oxidized products giving red and green fluorescence, respectively. Peculiar features of lipoperoxidation and BODIPY dye properties make this assessment a rather complex process, not exempt of doubts and troubles. Color changes and fluorescence fading that are not due to lipid peroxidation must be taken into account to avoid misleading results. As a characteristic feature of lipoperoxidation is the propagation of peroxyl radicals, pitfalls and advantages of a delayed detection by BODIPY probes should be considered.

Ixazomib, an oral proteasome inhibitor, exhibits potential effect in dystrophin-deficient mdx mice.

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm2 in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF-ß in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.

BODIPYs bearing a dimethylaminopropoxy substituent for imaging and photodynamic inactivation of bacteria.

A new BODIPY (BDP 1) bearing a dimethylaminopropoxy group attached to a phenylene unit was synthesized. This compound was brominated to obtain the halogenated analog BDP 2, which was designed to enhance the photodynamic effect of BODIPY to kill bacteria without an intrinsic cationic charge. The basic amino group located at the end of the propoxy bridge can acquire a positive charge by protonation in an aqueous medium, increasing the binding to bacterial cells. Interaction and photokilling activity mediated by these compounds was evaluated in Staphylococcus aureus and Escherichia coli. BDP 1 and BDP 2 were rapidly bound to bacterial cells, showing bioimages with green emission. Complete elimination of S. aureus was detected when cells were incubated with 1 µM BDP 2 and irradiated for 5 min. Comparable photoinactivation was obtained with E. coli, after an irradiation of 30 min. Furthermore, BDP 2 was effective to kill bacteria at very low concentration (0.5 µM). Thus, BDP 1 showed mainly interesting properties as a fluorophore, whereas BDP 2 was highly effective photosensitizer as a broad-spectrum antibacterial agent.

Inhibition of chymotrypsin-like activity of the proteasome by ixazomib prevents mitochondrial dysfunction during myocardial ischemia.

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

2-Aminoethyldiphenyl Borinate: A Multitarget Compound with Potential as a Drug Precursor.

BACKGROUND: Boron is considered a trace element that induces various effects in systems of the human body. However, each boron-containing compound exerts different effects. OBJECTIVE: To review the effects of 2-Aminoethyldiphenyl borinate (2-APB), an organoboron compound, on the human body, but also, its effects in animal models of human disease. METHODS: In this review, the information to showcase the expansion of these reported effects through interactions with several ion channels and other receptors has been reported. These effects are relevant in the biomedical and chemical fields due to the application of the reported data in developing therapeutic tools to modulate the functions of the immune, cardiovascular, gastrointestinal and nervous systems. RESULTS: Accordingly, 2-APB acts as a modulator of adaptive and innate immunity, including the production of cytokines and the migration of leukocytes. Additionally, reports show that 2-APB exerts effects on neurons, smooth muscle cells and cardiomyocytes, and it provides a cytoprotective effect by the modulation and attenuation of reactive oxygen species. CONCLUSION: The molecular pharmacology of 2-APB supports both its potential to act as a drug and the desirable inclusion of its moieties in new drug development. Research evaluating its efficacy in treating pain and specific maladies, such as immune, cardiovascular, gastrointestinal and neurodegenerative disorders, is scarce but interesting.

Benzoxaboroles as dynamic covalent receptors for bioconjugation and transport of nucleosides and related drugs: Proof of action in HeLa cells.

In this work we describe not previously explored binding studies on the reversible interaction of benzoxaborole with ligands of medical and pharmaceutical interest such as nucleosidic drugs gemcitabine and capecitabine, as well as the hydrophobic chemotherapeutic doxorubicin. We include functional derivatives of benzoxaborole such as a near infrared fluorescent boronolectine, Cy-Bx, The dynamic covalent interaction in physiological conditions was assessed by spectroscopic techniques yielding moderate to high binding affinities. The cytotoxic activity of the drugs upon conjugation to the boronolectins was evaluated revealing significant influence of the bioconjugation status on the cellular viability. The availability of the conjugate for cellular uptake and localization in the model cancer cell line HeLa was assessed by fluorescence imaging. Benzoxaborole and the fluorescent boronolectin Cy-Bx, proved to be versatile conjugation tools for 1,2 and 1,3-diol containing pharmacophores as well as bioisosteric forms such as 1,2-hydroxyamino, envisioning these small boronolectins as components in systems for drug release with tracking capability.

Light-Harvesting Antenna and Proton-Activated Photodynamic Effect of a Novel BODIPY-Fullerene C60 Dyad as Potential Antimicrobial Agent.

A covalently linked BODIPY-fullerene C60 dyad (BDP-C60 ) was synthesized as a two-segment structure, which consists of a visible light-harvesting antenna attached to an energy or electron acceptor moiety. This structure was designed to improve the photodynamic action of fullerene C60 to inactivate bacteria. The absorption spectrum of BDP-C60 was found to be a superposition of the spectra of its constitutional moieties, whereas the fluorescence emission of the BODIPY unit was strongly quenched by the fullerene C60 . Spectroscopic, calculations, and redox studies indicate a competence between photoinduced energy and electron transfer. Protonating the dimethylaminophenyl substituent through addition of an acidic medium led to a substantial increase in the fluorescence emission, triplet excited state formation, and singlet molecular oxygen production. At physiological pH, photosensitized inactivation of Staphylococcus aureus mediated by 1 µM BDP-C60 exhibited a 4.5 log decrease of cell survival (>99.997 %) after 15 min irradiation. A similar result was obtained with Escherichia coli using 30 min irradiation. Moreover, proton-activated photodynamic action of BDP-C60 turned this dyad into a highly effective photosensitizer to eradicate E. coli. Therefore, BDP-C60 is an interesting photosensitizing structure in which the light-harvesting antenna effect of the BODIPY unit combined with the protonation of dimethylaminophenyl group can be used to improve the photoinactivation of bacteria.

Insights into the Structural Requirements of 2(S)-Amino-6-Boronohexanoic Acid Derivatives as Arginase I Inhibitors: 3D-QSAR, Docking, and Interaction Fingerprint Studies.

Human arginase I (hARGI) is an important enzyme involved in the urea cycle; its overexpression has been associated to cardiovascular and cerebrovascular diseases. In the last years, several congeneric sets of hARGI inhibitors have been reported with possible beneficial roles for the cardiovascular system. At the same time, crystallographic data have been reported including hARGI⁻inhibitor complexes, which can be considered for the design of novel inhibitors. In this work, the structure⁻activity relationship (SAR) of Cα substituted 2(S)-amino-6-boronohexanoic acid (ABH) derivatives as hARGI inhibitors was studied by using a three-dimensional quantitative structure⁻activity relationships (3D-QSAR) method. The predictivity of the obtained 3D-QSAR model was demonstrated by using internal and external validation experiments. The best model revealed that the differential hARGI inhibitory activities of the ABH derivatives can be described by using steric and electrostatic fields; the local effects of these fields in the activity are presented. In addition, binding modes of the above-mentioned compounds inside the hARGI binding site were obtained by using molecular docking. It was found that ABH derivatives adopted the same orientation reported for ABH within the hARGI active site, with the substituents at Cα exposed to the solvent with interactions with residues at the entrance of the binding site. The hARGI residues involved in chemical interactions with inhibitors were identified by using an interaction fingerprints (IFPs) analysis.

Synthesis and Evaluation of the Anticancer and Trypanocidal Activities of Boronic Tyrphostins.

Molecules containing an (cyanovinyl)arene moiety are known as tyrphostins because of their ability to inhibit proteins from the tyrosine kinase family, an interesting target for the development of anticancer and trypanocidal drugs. In the present work, (E)-(cyanovinyl)benzeneboronic acids were synthesized by Knoevenagel condensations without the use of any catalysts in water through a simple protocol that completely avoided the use of organic solvents in the synthesis and workup process. The in vitro anticancer and trypanocidal activities of the synthesized boronic acids were also evaluated, and it was discovered that the introduction of the boronic acid functionality improved the activity of the boronic tyrphostins. In silico target fishing with the use of a chemogenomic approach suggested that tyrosine-phosphorylation-regulated kinase 1a (DYRK1A) was a potential target for some of the designed compounds.

Blocking IP3 signal transduction pathways inhibits melatonin-induced Ca2+ signals and impairs P. falciparum development and proliferation in erythrocytes.

Inositol 1,4,5 trisphosphate (IP3) signaling plays a crucial role in a wide range of eukaryotic processes. In Plasmodium falciparum, IP3 elicits Ca2+ release from intracellular Ca2+ stores, even though no IP3 receptor homolog has been identified to date. The human host hormone melatonin plays a key role in entraining the P. falciparum life cycle in the intraerythrocytic stages, apparently through an IP3-dependent Ca2+ signal. The melatonin-induced cytosolic Ca2+ ([Ca2+]cyt) increase and malaria cell cycle can be blocked by the IP3 receptor blocker 2-aminoethyl diphenylborinate (2-APB). However, 2-APB also inhibits store-operated Ca2+ entry (SOCE). Therefore, we have used two novel 2-APB derivatives, DPB162-AE and DPB163-AE, which are 100-fold more potent than 2-APB in blocking SOCE in mammalian cells, and appear to act by interfering with clustering of STIM proteins. In the present work we report that DPB162-AE and DPB163-AE block the [Ca2+]cyt rise in response to melatonin in P. falciparum, but only at high concentrations. These compounds also block SOCE in the parasite at similarly high concentrations suggesting that P. falciparum SOCE is not activated in the same way as in mammalian cells. We further find that DPB162-AE and DPB163-AE affect the development of the intraerythrocytic parasites and invasion of new red blood cells. Our efforts to episomally express proteins that compete with native IP3 receptor like IP3-sponge and an IP3 sensor such as IRIS proved to be lethal to P. falciparum during intraerythrocytic cycle. The present findings point to an important role of IP3-induced Ca2+ release in intraerythrocytic stage of P. falciparum.

Methylboronic acid fertilization alleviates boron deficiency symptoms in Arabidopsis thaliana.

MAIN CONCLUSION: Our results showed that methylboronic acid is capable of alleviating boron deficiency, enhancing plant growth, and is less toxic than boric acid at higher concentrations. Boron is an essential plant micronutrient and its deficiency occurs in several regions globally, resulting in impaired plant growth. Boron fertilization is a common agricultural practice, but the action range of boron is narrow, sharply transitioning from deficiency to toxicity. Boric acid (BA) is the most common chemical form used in agriculture. In this work, we describe that methylboronic acid (MBA) is capable of alleviating boron deficiency in Arabidopsis. MBA is a boronic acid, but does not naturally occur in soils, necessitating synthesis. Other boronic acids have been described as boron competitors in plants, inhibiting auxin biosynthesis and root development. MBA is more water-soluble than BA and delivers the same amount of boron per molecule. We observed that Arabidopsis seedlings grown in the presence of MBA presented higher numbers of lateral roots and greater main root length compared to plants grown in BA. In addition, root hair length and leaf surface area were increased using MBA as a boron fertilizer. Finally, MBA was less toxic than BA at high concentrations, producing a slight reduction in the main root length but no decrease in total chlorophyll. Our results open a new opportunity to explore the use of a synthetic form of boron in agriculture, providing a tool for future research for plant nutrition.

Single agent and synergistic combinatorial efficacy of first-in-class small molecule imipridone ONC201 in hematological malignancies.

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1-8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

Calcium dynamics in tomato pollen tubes using the Yellow Cameleon 3.6 sensor.

KEY MESSAGE: In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca2+]cyt gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca2+ channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.