Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model

Araceli Tobío; Amparo Alfonso; Iris Madera-Salcedo; Luis M. Botana; Ulrich Blank

doi:10.1371/journal.pone.0167572

Abstract

Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug.

Citation: Tobío A, Alfonso A, Madera-Salcedo I, Botana LM, Blank U (2016) Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model. PLoS ONE 11(12): e0167572. https://doi.org/10.1371/journal.pone.0167572

Editor: Antonio Facchiano, IDI, Istituto Dermopatico dell'Immacolata, ITALY

Received: June 1, 2016; Accepted: November 15, 2016; Published: December 14, 2016

Copyright: © 2016 Tobío et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: Dr. Araceli Tobio Ageitos was supported by a postdoctoral fellowship from Fundación Juana de Vega, Spain. Dr. Iris Madera-Salcedo was supported by an International collaboration grant between ANR France (ANR-12-ISV3-0006-01) and Conacyt Mexico (Conacyt-ANR 188565). This research project has been supported by the Investissements d’Avenir programme ANR-11-IDEX-0005-02, Sorbonne Paris Cite, Laboratoire d’excellence INFLAMEX, and DHU Fire. This work was also supported by the COST Action BM1007 (Mast cells and basophils–targets for innovative therapies) of the European Community.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: DNP, dinitrophenyl; MC, mast cell; RBL, rat basophilic leukemia; YTX, yessotoxin

Introduction

Yessotoxins (YTXs) are a group of lipophilic marine exotoxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera [1–3]. The group of YTXs is composed of close to 90 known analogs (YTX, homoYTX, hydroxyYTX, carboxyYTX and desulfoYTX among others) [4], however, the chemical structures for most of these compounds remain unclear [1]. Although no lethality was observed after oral administration in mice (doses up to 54mg/Kg), YTXs were reported to be lethal after intraperitoneal injection causing restlessness, dyspnoea, shivering, jumping and cramps albeit at relatively high levels of median lethal dose (LD₅₀) values ranging from 80 to 750μg/Kg depending on the mouse strain used [5]. YTX is the most toxic among all the analogous, nevertheless, the toxicity following intraperitoneal injection or oral administration at sublethal doses does not induce neither macroscopic nor histological alterations, even in the cardiac muscle [6, 7]. Another study, however, reports some moderate changes and damage in the myocardium that are reversible in the long-term [8, 9].

The role of YTX compounds as marine seafood contaminant toxins is presently debated. Based on the toxicity observed in mouse bioassays and their coexistence with other marine toxins (okadaic acid and dinophysistoxins) they have been included in the list of marine toxins [8]. While the European Food Safety Authority (EFSA) sets an Acute Reference Dose (ARfD) of 25μg YTX equivalents/Kg body weight, the European Union has established a limit of 3.75mg YTX/Kg of shellfish meat, as a prevention measure [10]. Yet, it remains a fact that the biological activity of YTXs in the environment is incompletely understood.

In order to better understand the mechanism of action of YTXs some of their activities have been studied in vitro in previous works. An intriguing finding revealed a cytotoxic effect of YTX described initially in rat glioma cells [11] and hepatocytes [12]. Subsequent studies performed in BE(2)-M17 neuroblastoma demonstrated that YTX induces apoptotic cell death [13]. Likewise, tumoural K-562 lymphocytes were shown to be YTX-sensitive dying by apoptosis, while normal human lymphocytes proved to be YTX-resistant [14, 15]. In some mouse cell lines autophagy activity was found after YTX exposure [16]. However, no in vivo studies testing the cytotoxic effects of YTX in the treatment of tumours have been reported so far.

In addition to cytotoxicity, anti-asthmatic and anti-allergic effects have been proposed for the YTX group, albeit these therapeutic effects are not completely understood [17, 18]. The interaction of allergens and allergen-specific IgE with the high-affinity IgE receptor (FcεRI) represents the key event in type I hypersensivity allergic reactions triggering a signalling cascade enabling rapid release of multiple inflammatory mediators such as histamine from mast cells (MCs) and basophils [19]. MCs are derived from CD34⁺/c-kit⁺ progenitor cells and play a crucial role in the pathogenesis of allergy, inflammation and many chronic inflammatory processes [20]. However, no studies examining directly the effect of YTX on the viability and cellular activation as well as the degranulation of MCs have been reported so far. We therefore sought to examine the activities of YTX on MC biology. Our results show that the anti-allergic effect is minor, while the toxin profoundly inhibits the growth of tumour MCs and some other tumour cells, which undergo apoptosis, while primary bone marrow derived MCs were YTX resistant. We also studied the cytotoxic properties of YTX in vivo using the well-established B16F10 melanoma model in mice to clarify the capacity of this molecule as an anti-cancer compound.

Materials and Methods

Reagents

YTX (purity ≥ 99%) dissolved in methanol was obtained from Cifga Laboratorio (Lugo, Spain). 4-Nitrophenyl N-acetyl-β-D-glucosaminide (pNAG), propidium iodide solution, MgCl₂, glucose, DNP-human serum albumin (DNP-HSA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (Germany). Annexin V-FITC and 10X AnnexinV Binding Buffer were from BD Pharmingen (France). HEPES was from Gibco (Life Technologies). NaCl was from VWR (Belgium). KCl and Triton X-100 were from Euromedex (France). CaCl₂ was from Prolano (Paris, France). Bovine serum albumin (BSA) was from PAA (Austria). Mouse monoclonal anti-DNP IgE has been described previously [21–23].

Animals

The C57/BL6 strain was raised and maintained at the animal facilities of the Center of Research on Inflammation. All experiments were performed in accordance with the national ethical guidelines and with the approval of local authorities of the Comité d’Éthique Expérimentation Animale Bichat-Debré. Animals were in cages with the following dimensions: 30x20x13 (LxWxH) and separated by three groups (Untreated, Vehicle and Yessotoxin group). Each cage was labeled with a card indicating the study, group and animal number. A maximum of 3 animals of the same group were housed in the same cage. Mice were maintained in a specific pathogen-free facility with a 12-h light/12-h dark cycle. Sterile food and sterilized pure water was provided ad libitum during the whole procedure.

Cells and cell lines

Bone marrow-derived mast cells (BMMCs) were derived from the bone marrow of C57BL/6 mice. The mice were sacrificed by CO₂ inhalation and bone marrow cells were cultured in IMDM containing 15% fetal bovine serum (FBS) (Gibco, Life Technologies), 1mM sodium pyruvate (GE Healthcare), 100μM non-essential amino acids (Gibco, Life Technologies), 100UI/mL penicillin, 100μg/mL streptomycin (Gibco, Life Technologies) and 0.05mM 2-mercaptoethanol ≥ 99% (Gibco, Life Technologies). The medium is supplemented with 10ng/mL interleukin-3 and 10ng/mL Stem Cell Factor (both from Miltenylbiotec). Cells were cultured in an atmosphere containing 5% CO₂. After five weeks in culture more than 95% of the cells were MCs assessed by toluidine blue staining.

RBL-2H3 and B16F10 melanoma (kindly provided by Dr. Thiago Maciel, Imagine Institute, Paris, France) cell lines were grown at 37°C in DMEM-Glutamax High Glucose medium (Gibco, Life Technologies) containing 10% FBS, 100UI/mL penicillin, 100μg/mL streptomycin (Gibco, Life Technologies) and for B16F10 additionally 1mM sodium pyruvate (Gibco, Life Technologies), 100μM non-essential amino acids and 0.05mM 2-mercaptoethanol ≥ 99% (Gibco, Life Technologies) in an atmosphere containing 5% CO₂. The MEC1 cell line (kindly provided by Dr. Pierre Launay, INSERM U1149, Paris, France) was maintained at 37°C in RPMI-Glutamax medium (Gibco, Life Technologies) supplemented with 10% FBS, 100UI/mL penicillin and 100μg/mL streptomycin (Gibco, Life Technologies) in an atmosphere containing 5% CO₂.

β-hexosaminidase release measurement

Cells (2x10⁶/mL) were incubated with monoclonal anti-DNP IgE ascites fluid (1:10,000 final dilution) overnight. After washing, cells were resuspended in 500μL Tyrode's buffer (20mM HEPES pH 7.2, 137mM NaCl, 5mM KCl, 1mM MgCl₂, 1,8mM CaCl₂, 5,6mM glucose, 0,5mg/mL BSA). Cells were then treated with indicated concentrations of YTX or vehicle (methanol) for 30 min and 1h before stimulation for 45min with DNP-HSA in the presence or absence of YTX as indicated. The reaction is stopped by immersing cells on ice for 10min. The percent released β-hexosaminidase into the supernatant was then determined as previously described [24], after subtracting background release of non-stimulated cells. 100% values were determined after addition of 0.5% Triton X-100 to lyse the cells.

Determination of cellular viability and apoptosis/necrosis

Cell viability was measured using the MTT test. This assay measures mitochondrial function by determining the quantity of formazan formed after conversion of the soluble MTT dye by active mitochondrial dehydrogenases in live cells. Briefly, after incubation with YTX cells (0.5x10⁶/mL) were washed in 500μL Tyrode's buffer before addition to MTT (250μg/mL) and incubation for 30min at 37°C [14, 25, 26]. After incubation cells were centrifuged and resuspended in 100μL H₂O and sonicated. Final optical density obtained from formazan formation was measured at 595nm with a multi-mode plate reader (Infinite M200, Tecan^®). Cells lysed in 10% Triton X-100 were used as 100% positive control for death cells.

Apoptosis and necrotic cell death were determined by flow cytometry using AnnexinV-FITC/propidium iodide (PI) staining. Cells (1x10⁶cells/mL) were incubated with different concentrations of YTX for indicated times. Cells were washed with Tyrode's buffer, resuspended in 100μL 1X AnnexinV Binding Buffer and stained by adding 5μL of AnnexinV-FITC (BD Pharmingen) for 15min at RT as well as of 2.5μg/mL PI solution (Sigma-Aldrich). Flow cytometry data were acquired on a Fortessa flow cytometer (BD Biosciences) and analysed using FlowJo software (TreeStar). Live cells were considered both Annexin V-FITC and PI negative; early apoptotic cells were considered Annexin V-FITC positive and PI negative, late apoptotic cells were considered both Annexin V-FITC and PI positive and necrotic cells were considered Annexin V-FITC negative and PI positive.

B16F10 mouse model of subcutaneous melanoma

Anti-cancer effect of YTX in vivo was studied using the B16F10 melanoma mouse model. A total of 2x10⁵ B16F10 melanoma cells in 100μL PBS were injected subcutaneously into the flank of C57BL/6 females (7 weeks) inducing within 5 to 10 days a palpable tumour of about 50mm³ [27]. At this time point, mice were treated with 100μg/Kg of YTX or vehicle administrated subcutaneously in close vicinity to the site of tumour development. Additional lower doses of YTX (20μg/Kg and 10μg/Kg) were administrated subcutaneously as indicated. As the B16 melanoma is a fast growing tumour, its volume was determined daily until sacrifice by the following formula for solid tumours: volume = (length x width²)/2 [28, 29]. Treatment time was kept to a minimum of 5 days and mice were euthanized by CO₂ inhalation (5 L/min) for 10 minutes at day 12 allowing a maximum of 7 days of tumour growth before they reached necrotic or ulcerating stages as previously recommended [27]. Signs like no interest on cage exploration, weight and/or appetite loss, difficulty in breathing and loss of coordination were evaluated two times per day. Additional signs of toxicity such us difficulty to eat, drink, walk or groom, distress or moribund signs or any other signs of systemic toxicity were also monitored for immediate euthanization. None of the animals suffered from any of these signs during the procedure, therefore, no animals were euthanized during the study. The maximal tumour volume achieved was in the range or lower of previously published data [30–34]. The weight loss at the day of sacrifice considering tumour density [35] was less than 20%. A blood sample was collected before sacrifice and immediately analysed in the Melet Schloesing Haematology Analyser for haematological parameters determination including lymphocytes, monocytes, neutrophils/granulocytes, erythrocytes and platelets. All experiments were performed in accordance with the national ethical guidelines and with the approval of local authorities of the Comité d’Éthique Expérimentation Animale Bichat-Debré.

Glucose uptake measurement in B16F10 mouse model of subcutaneous melanoma

At day of sacrifice 100μL of RediJect 2-DG Fluorescent Imaging Probe (Perkin Elmer^®) were injected intravenously to visualize glucose uptake known to be directly related to cell proliferation and hence tumour growth. After 3h, animals were anesthesized with 5mg Ketamine (Ketamine 50mg, Virbac^®) and 20mg Xilacin (Rompun 2%, Bayern^®) diluted in PBS and injected intraperitoneally. Luminiscence images were captured using a FX Pro (Kodak) and analyzed with Carestream MI software.

Statistical analysis

One-way ANOVA was employed for comparison of significant differences among groups. A probability level of 0.05 or smaller was used for statistical significance.

Results

To analyse whether the reported anti-allergic and anti-asthmatic activity of YTX involved its ability to inhibit MC degranulation we evaluated the effect of YTX on the release of granule-stored β-hexosaminidase in the RBL-2H3 MC line and in primary BMMCs. IgE-sensitized RBL-2H3 cells and BMMCs were treated with 10 or 30 nM YTX for 30 or 60min before stimulating them with specific antigen (DNP-HSA) for 45min in the presence or absence (removal) of YTX. In RBL-2H3 cells no significant effect was observed at low and high antigen doses (10ng/mL and 1000ng/mL) while at more optimal stimulatory doses (100ng/mL), a slight decrease in the degranulation response was observed at 30nM of YTX, particularly when the toxin remained present during the stimulation period (Fig 1A). A decrease of the degranulation response was also observed in BMMCs after 1h incubation of YTX, independently of whether it had been removed before stimulation or not (Fig 1B, right panel). The inhibitory effect required prolonged preincubation with YTX as it was only observed after pretreatment for 1h, while no effect was observed after 30min (Fig 1B, left panel). Although these results demonstrate that YTX decreases β-hexosaminidase release, its effect was not very potent reaching with a maximum inhibition of 25% in both BMMCs and RBL cells. It also appeared to be dependent on the dose of antigen in RBL-2H3 cells disappearing at non optimal stimulatory doses (Fig 1A).

Download:

Fig 1. Effect of YTX on β-hexosaminidase release induced by IgE/Ag in RBL-2H3 and BMMCs.

IgE-sensitized RBL-2H3 cells (A) or BMMCs (B) were incubated with 10 and 30nM YTX or vehicle (methanol) for 30 or 60min. Then YTX was removed (+) from the medium or not (-) and cells were stimulated for 45min with DNP-HSA antigen at the indicated concentrations. The percentage release of β-hexosaminidase was determined and compared to vehicle treated cells. No effect was observed after vehicle incubation. Data (percentage release) are the mean ± SEM of three experiments. Significant differences between DNP-HSA- (hatched) and DNP-HSA+YTX-treated cells: (*) p≤0.05.

https://doi.org/10.1371/journal.pone.0167572.g001

As YTX was shown previously to be toxic to tumour cells at relatively low concentrations, we performed control experiments to evaluate whether the toxin had any effect on MC viability. Fig 2 shows that using the MTT assay YTX did not induce any cytotoxic effect neither in BMMCs nor in RBL-2H3 after short (30min) incubation times. However, after 1h of incubation time a slight but significant decrease or tendency to decrease in cell viability was observed in RBL-2H3 cells at the high YTX concentrations (30, 50 and 100nM). This effect became highly prominent after 24, 48 and 72h suggesting that the toxin exhibited significant toxicity towards the RBL-2H3 MC cells line. Surprisingly, the effect was different when BMMCs were examined as no significant toxicity of YTX could be observed. Only with the highest concentration of YTX (100nM) a tendency for some toxicity was observed at 48h and 72h of treatment. Taken together, these results indicate that YTX has a clear cytotoxic effect for the MC tumour line, while primary MCs were highly resistant to YTX induced toxicity.

Download:

Fig 2. Effect of YTX on RBL-2H3 and BMMCs cell viability.

Cells were incubated with 10, 30, 50 and 100nM YTX for the indicated times and cell viability was assessed by the MTT assay. Corresponding controls with YTX solvent (methanol) were performed and cell viability was arbitrarily set to 100%. Of note, solvent did not significantly affect cell viability as compared to non-treated cells even at the highest concentration of vehicle. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.

https://doi.org/10.1371/journal.pone.0167572.g002

The pathway by which YTX induced cell death in RBL-2H3 cells remained unclear. We therefore used an Annexin V-FITC/PI staining assay to determine whether YTX-induced apoptosis or necrosis in these cells. The assay allowed to distinguish viable (Annexin V-FITC -/PI -), early apoptotic (Annexin V-FITC +/ PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +). Fig 3 shows, that upon treatment of RBL-2H3 cells with different doses of YTX the cells lost cell viability in a dose dependent manner undergoing a transition from early apoptosis after 24h to late apoptotic cells, which become highly prominent at the later time points of treatment. Some necrotic cells appeared at early time points and they significantly increased before 72h. The apoptosis induction is clearly dose dependent becoming rapidly apparent at 30nM, while the lower dose of 10nM only started to show some apoptotic effects at 72h, albeit this was not significant. Taken together these results indicate that YTX induces cell death by apoptosis.

Download:

Fig 3. YTX treatment induced apoptosis and necrosis in the RBL-2H3 cell line.

RBL-2H3 cells were either left untreated or were treated for indicated times with 10, 30, 50 and 100nM YTX. YTX solvent controls were performed and no effects were observed. Cells were then analysed for Annexin V-FITC and PI staining by flow cytometry and the percentage of live cells (Annexin V-FITC -/ PI -), early apoptotic (Annexin V-FITC +/PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +) was determined. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.

https://doi.org/10.1371/journal.pone.0167572.g003

The observed results corroborate previous works confirming the anti-tumour activity of YTX [14, 15]. In order to study the most suitable cell line for the in vivo experiment we selected two different cell lines, MEC1 and B16F10, both of which have been used as a tumour in vivo mouse model in our laboratory. MEC1 is a human cell line obtained from a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation [36], whereas B16F10 is a mouse melanoma cell line [27]. The cytotoxic effect of YTX treatment was studied in both cell lines using the same kinetics as above for RBL-2H3 cells. Fig 4A shows, that YTX induces a significant decrease in cell viability at 24h of treatment at 10, 30 and 100nM doses, in MEC1 cells. The cytotoxic effect of YTX was even stronger after 48h causing a significant decrease in cell viability (around 50%) at all YTX concentrations but cytotoxicity did not decrease further at 72h of incubation with YTX. For B16F10 cells the results show that YTX caused a progressive decrease in cell viability with essentially no remaining viable cells after 72h of incubation with YTX (Fig 4B). This effect was observed at all treatment doses studied. Even the smallest dose decreased viability by up to 90%. These results were confirmed when cells were stained with Annexin V-FITC and PI (Fig 5). Results showed that already after 24h a significant proportion exhibited early signs of apoptosis and that this proportion increased significantly at later time points.

Download:

Fig 4. Effect of YTX on MEC1 and B16F10 cell line viability.

The MEC1 and B16F10 cell lines were incubated with 10, 30, 50 and 100nM YTX for the indicated times and cell viability was assessed by the MTT assay at the indicated time points. Corresponding controls with YTX solvent were performed and cell viability was arbitrarily set to 100%. Of note, solvent did not significantly affect cell viability as compared to non-treated cells even at the highest concentration of vehicle. Data are the mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.

https://doi.org/10.1371/journal.pone.0167572.g004

Download:

Fig 5. YTX treatment induced apoptosis in B16F10 cells.

B16F10 cells were either left untreated or were treated for indicated times with 10, 30, 50 and 100nM YTX. YTX solvent controls were performed and no effects were observed. Cells were then analyzed for Annexin-V and PI staining by flow cytometry and the percentage of live cells (Annexin V-FITC -/PI -), early apoptotic (Annexin V-FITC +/PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +) was determined. Mean ± SEM of three experiments. Significant differences between untreated and YTX-treated cells: (*) p≤0.05, (**) p≤0.01 and (***) p≤0.001.

https://doi.org/10.1371/journal.pone.0167572.g005

The above results indicate that YTX induces a stronger cytotoxicity towards B16F10 cells as compared to the MEC1 cell line. We therefore selected the B16F10 mouse tumour model to test the YTX anti-tumour effect in vivo. To this end B16F10 cells were injected subcutaneously into the flanks of the mice and allowed to grow until appearance of a small palpable tumour bleb with a size of about 50 mm³. Tumour treatment was started with an initial injection at high dose (100μg/Kg) in immediate tumour vicinity followed by 4 injections at lower doses at days 2, 3, 4 and 5. Fig 6A and 6B show that YTX treatment caused a clear decrease in tumour volume. This decrease was significant reducing the volume of the tumour by more than 12 times at day 10. Moreover, tumour volume did not differ between control and vehicle treated mice. Results obtained for the tumour weight after sacrifice corroborate the data observed in vivo with a significant decrease in tumour weight, whereas the treatment with vehicle does not have any effect (Fig 6C). With regard to the weight of the animals, no effect was observed neither after YTX nor vehicle treatment (Fig 6D) suggesting that such treatment was not particularly toxic for the mice. Furthermore, an analysis of haematological parameters was performed before sacrifice. Results obtained showed that YTX does not alter neither the percentage of white blood cells (lymphocytes, monocytes and neutrophils/granulocytes) (Fig 6E), nor erythrocytes (Fig 6F), nor platelets (Fig 6G). Supporting again the low toxicity of YTX when used for such treatment.

Download:

Fig 6. YTX treatment decreases tumour development in the B16F10 melanoma mouse model.

(A) Mice were injected subcutaneous B16F10 melanoma cells and tumours were allowed to develop until they reached a volume of 50 mm³ achieved between days 5 and 10. Mice were then either left untreated or were treated with the indicated concentrations of YTX or vehicle, which was injected subcutaneous right next to the tumour. (B) Representative glucose uptake after FX Pro Kodak image analysis of untreated, vehicle and YTX-treated mice at the day of sacrifice. (C) Corresponding resected tumour weight of animals after sacrifice at day 12 post-1st treatment. (D) Corresponding weight of untreated, vehicle- and YTX-treated mice. (E-G) Results of the haematological analysis performed before mice sacrifice. Data are expressed as mean ± SEM. Significant differences between untreated and YTX: (*) p≤0.05 and (***) p≤0.001.

https://doi.org/10.1371/journal.pone.0167572.g006

Discussion

The interest in medical applications for algal toxins and in particular of YTX due to its low oral toxicity has grown in recent years. One of the first potential roles discovered was the anti-allergic role of this compound [37]. This could relate to the capacity of YTX to activate cellular phosphodiesterases (PDEs known to play a role in the activation of basophils and MCs) [17, 38, 39]. As no systematic evaluation on the capacity of YTX on MC degranulation has been reported we studied the YTX effect on MCs degranulation using primary BMMCs and the RBL-2H3 cell line. Our results demonstrate that although YTX causes a significant decrease of β-hexosaminidase release, this effect appears minor reaching a maximum of 25% inhibition in BMMCs and RBL-2H3 cells being observed in the RBL-2H3 MC line only at optimal antigen doses.

Furthermore, the present paper also demonstrated that YTX is a secondary metabolite showing an important cytotoxic effect towards the RBL-2H3 cell line at low doses in the 30 to 100nM range. Interestingly non-tumoural MCs show an extraordinary resistance as no important cytotoxic effects were observed at doses of up to 50 nM with some cytotoxicity becoming apparent at 100nM, but only after long-term incubation (72h). In addition to RBL-2H3 cells YTX exhibited also a high cytotoxic activity against MEC1 and B16F10 cell lines confirming several other studies including a National Cancer Institute human tumour cell line screen, which showed high toxicity to many tumour cell lines in the nanomolar range [18]. In particular, melanoma cell lines were the most sensitive to YTX, followed by lung, colon, leukemia, mammary, ovarian, central nervous system and renal cell lines [4, 18]. On the other hand, low toxicity was generally observed in primary cells. Thus, previous studies in fresh human lymphocytes and K-562 lymphoma cells showed that they were, respectively, resistant and sensitive to YTX [14, 15]. Differential effects of YTX also showed that it does not induce cell death features in a lymphoblastoid cell line, whereas it induced apoptosis in the lymphocytic cell line K-562 [40].

Three different cell death mechanisms can be induced by YTX. The apoptotic pathway including caspase 3 activation was first described in the BE(2)-M17 neuroblastoma cell line [13], whereas paraptosis (BC3H1 myoblast cells) [41] and autophagy (human glioma and K-562 cells) were more recently described [15, 41, 42]. Previous work also described YTX apoptosis induction in K-562 cell line (other by activating caspase 3 and 8 (extrinsic apoptotic pathway hallmark) and decreasing anti-apoptotic Bcl-2 protein levels [15]. In our present manuscript the apoptotic cell death pathway was found to be the major cause of death in RBL-2H3 or B16F10 cell lines. Indeed, YTX rapidly induces exposure of phosphatidylserine as one of the early signs of apoptosis ending in a late apoptotic stage characterized by the uptake of PI in RBL-2H3 cell line. These data suggest, while YTX can induce multiple cell death pathways, including apoptosis and the recently described paraptosis and autophagy, the apoptosis pathway seems to be the primary pathway in RBL2H3 and B16F10 cells.

Among three cell lines tested in the present study, B16F10 was shown to exhibit the highest sensitivity to YTX, followed by RBL-2H3 and MEC1. The B16F10 is a very aggressive murine melanoma cell line [43] and its high YTX sensitivity is in accordance with the results obtained in a previous wide scale screening, which showed high sensitivity of human melanoma cell lines to the compound [4]. Malignant melanoma is the sixth most common cancer in the US [27] calling for novel drugs/therapies to effectively treat melanoma patients. Cutaneous melanomas are generated from skin melanocytes and albeit recent progress has been made through the use of checkpoint inhibitors, the effectiveness of the treatment is highly variable and depends on the patient [27, 44–46]. Based on the significant in vitro efficacy of YTX in inducing cell death of B16F10 melanoma cells and the fact that YTX has never been evaluated as an anti-cancer drug in vivo we examined its efficacy in vivo in the B16F10 mouse melanoma model. Our results showed that local subcutaneous administration of YTX in the close vicinity to established tumour bleb resulted in a marked reduction in melanoma growth. One high dose bolus injection of 100μg/Kg followed by 4 additional low dose injections (20 and 10μg/Kg) were sufficient to reduce by 82% the B16F10 tumour melanoma volume after 12 days without affecting animal weight and haematological parameters. These results are encouraging and can be compared with other local therapeutic strategies tested in B16F10 melanoma murine model. Firstly, YTX in vitro cytotoxicity in B16F10 cells is 1000-fold more potent than the observed with the H-15 cyclic pentapeptide [47]. Another comparison includes the COOH-Terminal Peptide of Platelet Factor-4 Variant (CXCL4L1/PF-4var^47-70), which when injected intra-tumourally was also described as a successful therapy in B16F10 melanoma growth in vivo [48]. Tumour growth inhibition due to CXCL4L1/PF-4var^47-70 after 16 days treatment was about 60% but treatment started already at the time of inoculation with tumour cells. YTX treatment seems equivalent to therapeutic effects achieved with subcutaneous vaccination of a viral cocktail expressing a C-terminal peptide of human telomerase transcriptase, an enzyme expressed in more than 85% of human tumour cells but rarely in normal cells [33]. Therefore, the present work demonstrates, for first time, the activity of YTX as an anticancer compound in vivo making it a compound with a great interest in cancer therapy.

Author Contributions

Conceptualization: AT AA LMB UB.
Data curation: AT.
Formal analysis: AT.
Funding acquisition: AT AA LMB UB.
Investigation: AT AA LMB UB.
Methodology: AT IMS.
Project administration: AT UB.
Resources: AT UB.
Software: AT.
Supervision: AA LMB UB.
Validation: AT.
Visualization: AT UB.
Writing – original draft: AT.
Writing – review & editing: AT UB.

References

1. Satake M, MacKenzie L, Yasumoto T. Identification of Protoceratium reticulatum as the biogenetic origin of yessotoxin. Nat Toxins. 1997;5(4):164–7. Epub 1997/01/01. pmid:9407560
- View Article
- PubMed/NCBI
- Google Scholar
2. Draisci R, Ferretti E, Palleschi L, Marchiafava C, Poletti R, Milandri A, et al. High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea. Toxicon. 1999;37(8):1187–93. pmid:10400302
- View Article
- PubMed/NCBI
- Google Scholar
3. Pistocchi R, Guerrini F, Pezzolesi L, Riccardi M, Vanucci S, Ciminiello P, et al. Toxin levels and profiles in microalgae from the north-Western Adriatic Sea—15 years of studies on cultured species. Mar Drugs. 2012;10(1):140–62. pmid:22363227
- View Article
- PubMed/NCBI
- Google Scholar
4. Botana LM, Alfonso A, Vale C, Vilariño N, Rubiolo J, Alonso E, et al. The Mechanistic Complexities of Phycotoxins: Toxicology of Azaspiracids and Yessotoxins. Advances in Molecular Toxicology. 2014;8:1.
- View Article
- Google Scholar
5. Tubaro A, Dell'ovo V, Sosa S, Florio C. Yessotoxins: a toxicological overview. Toxicon. 2010;56(2):163–72. Epub 2009/08/08. pmid:19660487
- View Article
- PubMed/NCBI
- Google Scholar
6. Tubaro A, Sosa S, Carbonatto M, Altinier G, Vita F, Melato M, et al. Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon. 2003;41(7):783–92. Epub 2003/06/05. pmid:12782078
- View Article
- PubMed/NCBI
- Google Scholar
7. Tubaro A, Sosa S, Altinier G, Soranzo MR, Satake M, Della Loggia R, et al. Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice. Toxicon. 2004;43(4):439–45. Epub 2004/03/31. pmid:15051408
- View Article
- PubMed/NCBI
- Google Scholar
8. Aune T, Sorby R, Yasumoto T, Ramstad H, Landsverk T. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon. 2002;40(1):77–82. pmid:11602282
- View Article
- PubMed/NCBI
- Google Scholar
9. Tubaro A, Giangaspero A, Ardizzone M, Soranzo MR, Vita F, Yasumoto T, et al. Ultrastructural damage to heart tissue from repeated oral exposure to yessotoxin resolves in 3 months. Toxicon. 2008;51(7):1225–35. Epub 2008/04/12. pmid:18402996
- View Article
- PubMed/NCBI
- Google Scholar
10. Alexander J, Benford D, Boobis A, Ceccatelli S, Cravedi J-P, Di A, et al. Marine biotoxins in shellfish–Summary on regulated marine biotoxins Scientific Opinion of the Panel on Contaminants in the Food Chain. The EFSA Journal. 2009;1306:1–23.
- View Article
- Google Scholar
11. Ogino H, Kumagai M, Yasumoto T. Toxicologic evaluation of yessotoxin. Nat Toxins. 1997;5(6):255–9. pmid:9615314
- View Article
- PubMed/NCBI
- Google Scholar
12. Aune T. Toxicity of marine and freshwater algal biotoxins towards freshly prepared hepatocytes. Mycotoxins and phycotoxins. 1989;88:461–8.
- View Article
- Google Scholar
13. Leira F, Alvarez C, Vieites JM, Vieytes MR, Botana LM. Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 neuroblastoma cell line. Toxicol In Vitro. 2002;16(1):23–31. Epub 2002/01/29. pmid:11812636
- View Article
- PubMed/NCBI
- Google Scholar
14. Tobio A, Fernandez-Araujo A, Alfonso A, Botana LM. Role of yessotoxin in calcium and cAMP-crosstalks in primary and K-562 human lymphocytes: The effect is mediated by anchor kinase A mitochondrial proteins. J Cell Biochem. 2012. Epub 2012/07/19.
- View Article
- Google Scholar
15. Fernandez-Araujo A, Tobio A, Alfonso A, Botana LM. Role of AKAP 149-PKA-PDE4A complex in cell survival and cell differentiation processes. Int J Biochem Cell Biol. 2014;53C:89–101. Epub 2014/05/13.
- View Article
- Google Scholar
16. Korsnes MS, Kolstad H, Kleiveland CR, Korsnes R, Ormen E. Autophagic activity in BC3H1 cells exposed to yessotoxin. Toxicol In Vitro. 2015.
- View Article
- Google Scholar
17. Alfonso A, de la Rosa L, Vieytes MR, Yasumoto T, Botana LM. Yessotoxin, a novel phycotoxin, activates phosphodiesterase activity. Effect of yessotoxin on cAMP levels in human lymphocytes. Biochem Pharmacol. 2003;65(2):193–208. Epub 2002/12/31. pmid:12504795
- View Article
- PubMed/NCBI
- Google Scholar
18. Alfonso A, Vieytes MR, Botana LM. Yessotoxin, a Promising Therapeutic Tool. Mar Drugs. 2016;14(2).
- View Article
- Google Scholar
19. Blank U, Rivera J. The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol. 2004;25(5):266–73. Epub 2004/04/22. pmid:15099567
- View Article
- PubMed/NCBI
- Google Scholar
20. Galli SJ. The Mast Cell-IgE Paradox: From Homeostasis to Anaphylaxis. Am J Pathol. 2016;186(2):212–24. pmid:26776074
- View Article
- PubMed/NCBI
- Google Scholar
21. Martin-Verdeaux S, Pombo I, Iannascoli B, Roa M, Varin-Blank N, Rivera J, et al. Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis. J Cell Sci. 2003;116(Pt 2):325–34. pmid:12482918
- View Article
- PubMed/NCBI
- Google Scholar
22. Tiwari N, Wang CC, Brochetta C, Ke G, Vita F, Qi Z, et al. VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways. Blood. 2008;111(7):3665–74. pmid:18203950
- View Article
- PubMed/NCBI
- Google Scholar
23. Pfirsch-Maisonnas S, Aloulou M, Xu T, Claver J, Kanamaru Y, Tiwari M, et al. Inhibitory ITAM signaling traps activating receptors with the phosphatase SHP-1 to form polarized "inhibisome" clusters. Sci Signal. 2011;4(169):ra24. pmid:21505186
- View Article
- PubMed/NCBI
- Google Scholar
24. Paumet F, Le Mao J, Martin S, Galli T, David B, Blank U, et al. Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment. J Immunol. 2000;164(11):5850–7. pmid:10820264
- View Article
- PubMed/NCBI
- Google Scholar
25. Tobio A, Alfonso A, Botana LM. PKC Potentiates Tyrosine Kinase Inhibitors STI571 and Dasatinib Cytotoxic Effect. Anticancer Res. 2014;34(7):3347–56. Epub 2014/07/02. pmid:24982339
- View Article
- PubMed/NCBI
- Google Scholar
26. Tobio A, Alfonso A, Botana LM. C-kit mutations determine dasatinib mechanism of action in HMC-1 neoplastic mast cells: dasatinib differently regulates PKCdelta translocation in HMC-1(560) and HMC-1(560,816) cell lines. Immunopharmacol Immunotoxicol. 2015;37(4):380–7. pmid:26181649
- View Article
- PubMed/NCBI
- Google Scholar
27. Overwijk WW, Restifo NP. B16 as a mouse model for human melanoma. Curr Protoc Immunol. 2001;Chapter 20:Unit 20 1. pmid:18432774
- View Article
- PubMed/NCBI
- Google Scholar
28. Sos ML, Levin RS, Gordan JD, Oses-Prieto JA, Webber JT, Salt M, et al. Oncogene mimicry as a mechanism of primary resistance to BRAF inhibitors. Cell Rep. 2014;8(4):1037–48. pmid:25127139
- View Article
- PubMed/NCBI
- Google Scholar
29. Xia S, Lu Y, Wang J, He C, Hong S, Serhan CN, et al. Melanoma growth is reduced in fat-1 transgenic mice: impact of omega-6/omega-3 essential fatty acids. Proc Natl Acad Sci U S A. 2006;103(33):12499–504. pmid:16888035
- View Article
- PubMed/NCBI
- Google Scholar
30. Lee SH, Koo BS, Park SY, Kim YM. Anti-angiogenic effects of resveratrol in combination with 5-fluorouracil on B16 murine melanoma cells. Mol Med Rep. 2015;12(2):2777–83. pmid:25936796
- View Article
- PubMed/NCBI
- Google Scholar
31. Kobayashi T, Hamaguchi Y, Hasegawa M, Fujimoto M, Takehara K, Matsushita T. B cells promote tumor immunity against B16F10 melanoma. Am J Pathol. 2014;184(11):3120–9. pmid:25173132
- View Article
- PubMed/NCBI
- Google Scholar
32. Yang LP, Cheng P, Peng XC, Shi HS, He WH, Cui FY, et al. Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma. J Exp Clin Cancer Res. 2009;28:75. pmid:19500366
- View Article
- PubMed/NCBI
- Google Scholar
33. Huo L, Yao H, Wang X, Wong GW, Kung HF, Lin MC. Inhibition of melanoma growth by subcutaneous administration of hTERTC27 viral cocktail in C57BL/6 mice. PLoS One. 2010;5(9):e12705. pmid:20856939
- View Article
- PubMed/NCBI
- Google Scholar
34. Talebian F, Liu JQ, Liu Z, Khattabi M, He Y, Ganju R, et al. Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions. PLoS One. 2012;7(2):e31442. pmid:22319630
- View Article
- PubMed/NCBI
- Google Scholar
35. Baniyash M, Netanel T, Witz IP. Differences in cell density associated with differences in lung-colonizing ability of B16 melanoma cells. Cancer Res. 1981;41(2):433–7. pmid:7448787
- View Article
- PubMed/NCBI
- Google Scholar
36. Stacchini A, Aragno M, Vallario A, Alfarano A, Circosta P, Gottardi D, et al. MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation. Leuk Res. 1999;23(2):127–36. pmid:10071128
- View Article
- PubMed/NCBI
- Google Scholar
37. Botana LLM, Alfonso RA, Rodriguez VM, Loza GMI. Use of yessotoxin in the treatment of allergic and asthmatic processes. Google Patents; 2011.
38. Essayan DM. Cyclic nucleotide phosphodiesterases. J Allergy Clin Immunol. 2001;108(5):671–80. pmid:11692087
- View Article
- PubMed/NCBI
- Google Scholar
39. Botana LM, MacGlashan DW. Differential effects of cAMP-elevating drugs on stimulus-induced cytosolic calcium changes in human basophils. J Leukoc Biol. 1994;55(6):798–804. Epub 1994/06/01. pmid:7515096
- View Article
- PubMed/NCBI
- Google Scholar
40. Fernandez-Araujo A, Sanchez JA, Alfonso A, Vieytes MR, Botana LM. Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines. Front Pharmacol. 2015;6:124. pmid:26136685
- View Article
- PubMed/NCBI
- Google Scholar
41. Korsnes MS, Espenes A, Hetland DL, Hermansen LC. Paraptosis-like cell death induced by yessotoxin. Toxicol In Vitro. 2011;25(8):1764–70. pmid:21945047
- View Article
- PubMed/NCBI
- Google Scholar
42. Fernandez-Araujo A, Alfonso A, Vieytes MR, Botana LM. Key role of phosphodiesterase 4A (PDE4A) in autophagy triggered by yessotoxin. Toxicology. 2015. Epub 2015/01/13.
- View Article
- Google Scholar
43. Lai YW, Wang SW, Chang CH, Liu SC, Chen YJ, Chi CW, et al. Butein inhibits metastatic behavior in mouse melanoma cells through VEGF expression and translation-dependent signaling pathway regulation. BMC Complement Altern Med. 2015;15(1):445.
- View Article
- Google Scholar
44. Becker JC, Houben R, Schrama D, Voigt H, Ugurel S, Reisfeld RA. Mouse models for melanoma: a personal perspective. Exp Dermatol. 2010;19(2):157–64. pmid:19849715
- View Article
- PubMed/NCBI
- Google Scholar
45. Johnson DB, Sosman JA. Update on the targeted therapy of melanoma. Curr Treat Options Oncol. 2013;14(2):280–92. pmid:23420410
- View Article
- PubMed/NCBI
- Google Scholar
46. Wang DY, Sosman JA, Johnson DB. Combination Immunotherapy: An Emerging Paradigm in Cancer Therapeutics. Oncology (Williston Park). 2015;29(12).
- View Article
- Google Scholar
47. Liu Y, Zhang G, Wang H, Liu S, Chen J, Zhao L, et al. Novel cyclic pentapeptide H-15 induces differentiation and inhibits proliferation in murine melanoma B16 cells. Oncol Lett. 2016;11(2):1251–5. pmid:26893727
- View Article
- PubMed/NCBI
- Google Scholar
48. Vandercappellen J, Liekens S, Bronckaers A, Noppen S, Ronsse I, Dillen C, et al. The COOH-terminal peptide of platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly inhibits angiogenesis and suppresses B16 melanoma growth in vivo. Mol Cancer Res. 2010;8(3):322–34. pmid:20215425
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Satake M, MacKenzie L, Yasumoto T. Identification of Protoceratium reticulatum as the biogenetic origin of yessotoxin. Nat Toxins. 1997;5(4):164–7. Epub 1997/01/01. pmid:9407560
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Draisci R, Ferretti E, Palleschi L, Marchiafava C, Poletti R, Milandri A, et al. High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea. Toxicon. 1999;37(8):1187–93. pmid:10400302
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Pistocchi R, Guerrini F, Pezzolesi L, Riccardi M, Vanucci S, Ciminiello P, et al. Toxin levels and profiles in microalgae from the north-Western Adriatic Sea—15 years of studies on cultured species. Mar Drugs. 2012;10(1):140–62. pmid:22363227
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Botana LM, Alfonso A, Vale C, Vilariño N, Rubiolo J, Alonso E, et al. The Mechanistic Complexities of Phycotoxins: Toxicology of Azaspiracids and Yessotoxins. Advances in Molecular Toxicology. 2014;8:1.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref5] 5. Tubaro A, Dell'ovo V, Sosa S, Florio C. Yessotoxins: a toxicological overview. Toxicon. 2010;56(2):163–72. Epub 2009/08/08. pmid:19660487
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Tubaro A, Sosa S, Carbonatto M, Altinier G, Vita F, Melato M, et al. Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon. 2003;41(7):783–92. Epub 2003/06/05. pmid:12782078
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Tubaro A, Sosa S, Altinier G, Soranzo MR, Satake M, Della Loggia R, et al. Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice. Toxicon. 2004;43(4):439–45. Epub 2004/03/31. pmid:15051408
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Aune T, Sorby R, Yasumoto T, Ramstad H, Landsverk T. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon. 2002;40(1):77–82. pmid:11602282
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Tubaro A, Giangaspero A, Ardizzone M, Soranzo MR, Vita F, Yasumoto T, et al. Ultrastructural damage to heart tissue from repeated oral exposure to yessotoxin resolves in 3 months. Toxicon. 2008;51(7):1225–35. Epub 2008/04/12. pmid:18402996
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Alexander J, Benford D, Boobis A, Ceccatelli S, Cravedi J-P, Di A, et al. Marine biotoxins in shellfish–Summary on regulated marine biotoxins Scientific Opinion of the Panel on Contaminants in the Food Chain. The EFSA Journal. 2009;1306:1–23.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref11] 11. Ogino H, Kumagai M, Yasumoto T. Toxicologic evaluation of yessotoxin. Nat Toxins. 1997;5(6):255–9. pmid:9615314
View Article
PubMed/NCBI
Google Scholar

[40] View Article

[41] PubMed/NCBI

[42] Google Scholar

[ref12] 12. Aune T. Toxicity of marine and freshwater algal biotoxins towards freshly prepared hepatocytes. Mycotoxins and phycotoxins. 1989;88:461–8.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref13] 13. Leira F, Alvarez C, Vieites JM, Vieytes MR, Botana LM. Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 neuroblastoma cell line. Toxicol In Vitro. 2002;16(1):23–31. Epub 2002/01/29. pmid:11812636
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Tobio A, Fernandez-Araujo A, Alfonso A, Botana LM. Role of yessotoxin in calcium and cAMP-crosstalks in primary and K-562 human lymphocytes: The effect is mediated by anchor kinase A mitochondrial proteins. J Cell Biochem. 2012. Epub 2012/07/19.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref15] 15. Fernandez-Araujo A, Tobio A, Alfonso A, Botana LM. Role of AKAP 149-PKA-PDE4A complex in cell survival and cell differentiation processes. Int J Biochem Cell Biol. 2014;53C:89–101. Epub 2014/05/13.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref16] 16. Korsnes MS, Kolstad H, Kleiveland CR, Korsnes R, Ormen E. Autophagic activity in BC3H1 cells exposed to yessotoxin. Toxicol In Vitro. 2015.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref17] 17. Alfonso A, de la Rosa L, Vieytes MR, Yasumoto T, Botana LM. Yessotoxin, a novel phycotoxin, activates phosphodiesterase activity. Effect of yessotoxin on cAMP levels in human lymphocytes. Biochem Pharmacol. 2003;65(2):193–208. Epub 2002/12/31. pmid:12504795
View Article
PubMed/NCBI
Google Scholar

[60] View Article

[61] PubMed/NCBI

[62] Google Scholar

[ref18] 18. Alfonso A, Vieytes MR, Botana LM. Yessotoxin, a Promising Therapeutic Tool. Mar Drugs. 2016;14(2).
View Article
Google Scholar

[64] View Article

[65] Google Scholar

[ref19] 19. Blank U, Rivera J. The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol. 2004;25(5):266–73. Epub 2004/04/22. pmid:15099567
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref20] 20. Galli SJ. The Mast Cell-IgE Paradox: From Homeostasis to Anaphylaxis. Am J Pathol. 2016;186(2):212–24. pmid:26776074
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref21] 21. Martin-Verdeaux S, Pombo I, Iannascoli B, Roa M, Varin-Blank N, Rivera J, et al. Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis. J Cell Sci. 2003;116(Pt 2):325–34. pmid:12482918
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref22] 22. Tiwari N, Wang CC, Brochetta C, Ke G, Vita F, Qi Z, et al. VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways. Blood. 2008;111(7):3665–74. pmid:18203950
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref23] 23. Pfirsch-Maisonnas S, Aloulou M, Xu T, Claver J, Kanamaru Y, Tiwari M, et al. Inhibitory ITAM signaling traps activating receptors with the phosphatase SHP-1 to form polarized "inhibisome" clusters. Sci Signal. 2011;4(169):ra24. pmid:21505186
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref24] 24. Paumet F, Le Mao J, Martin S, Galli T, David B, Blank U, et al. Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment. J Immunol. 2000;164(11):5850–7. pmid:10820264
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref25] 25. Tobio A, Alfonso A, Botana LM. PKC Potentiates Tyrosine Kinase Inhibitors STI571 and Dasatinib Cytotoxic Effect. Anticancer Res. 2014;34(7):3347–56. Epub 2014/07/02. pmid:24982339
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref26] 26. Tobio A, Alfonso A, Botana LM. C-kit mutations determine dasatinib mechanism of action in HMC-1 neoplastic mast cells: dasatinib differently regulates PKCdelta translocation in HMC-1(560) and HMC-1(560,816) cell lines. Immunopharmacol Immunotoxicol. 2015;37(4):380–7. pmid:26181649
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref27] 27. Overwijk WW, Restifo NP. B16 as a mouse model for human melanoma. Curr Protoc Immunol. 2001;Chapter 20:Unit 20 1. pmid:18432774
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref28] 28. Sos ML, Levin RS, Gordan JD, Oses-Prieto JA, Webber JT, Salt M, et al. Oncogene mimicry as a mechanism of primary resistance to BRAF inhibitors. Cell Rep. 2014;8(4):1037–48. pmid:25127139
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref29] 29. Xia S, Lu Y, Wang J, He C, Hong S, Serhan CN, et al. Melanoma growth is reduced in fat-1 transgenic mice: impact of omega-6/omega-3 essential fatty acids. Proc Natl Acad Sci U S A. 2006;103(33):12499–504. pmid:16888035
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref30] 30. Lee SH, Koo BS, Park SY, Kim YM. Anti-angiogenic effects of resveratrol in combination with 5-fluorouracil on B16 murine melanoma cells. Mol Med Rep. 2015;12(2):2777–83. pmid:25936796
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref31] 31. Kobayashi T, Hamaguchi Y, Hasegawa M, Fujimoto M, Takehara K, Matsushita T. B cells promote tumor immunity against B16F10 melanoma. Am J Pathol. 2014;184(11):3120–9. pmid:25173132
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref32] 32. Yang LP, Cheng P, Peng XC, Shi HS, He WH, Cui FY, et al. Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma. J Exp Clin Cancer Res. 2009;28:75. pmid:19500366
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref33] 33. Huo L, Yao H, Wang X, Wong GW, Kung HF, Lin MC. Inhibition of melanoma growth by subcutaneous administration of hTERTC27 viral cocktail in C57BL/6 mice. PLoS One. 2010;5(9):e12705. pmid:20856939
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref34] 34. Talebian F, Liu JQ, Liu Z, Khattabi M, He Y, Ganju R, et al. Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions. PLoS One. 2012;7(2):e31442. pmid:22319630
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref35] 35. Baniyash M, Netanel T, Witz IP. Differences in cell density associated with differences in lung-colonizing ability of B16 melanoma cells. Cancer Res. 1981;41(2):433–7. pmid:7448787
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref36] 36. Stacchini A, Aragno M, Vallario A, Alfarano A, Circosta P, Gottardi D, et al. MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation. Leuk Res. 1999;23(2):127–36. pmid:10071128
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref37] 37. Botana LLM, Alfonso RA, Rodriguez VM, Loza GMI. Use of yessotoxin in the treatment of allergic and asthmatic processes. Google Patents; 2011.

[ref38] 38. Essayan DM. Cyclic nucleotide phosphodiesterases. J Allergy Clin Immunol. 2001;108(5):671–80. pmid:11692087
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref39] 39. Botana LM, MacGlashan DW. Differential effects of cAMP-elevating drugs on stimulus-induced cytosolic calcium changes in human basophils. J Leukoc Biol. 1994;55(6):798–804. Epub 1994/06/01. pmid:7515096
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref40] 40. Fernandez-Araujo A, Sanchez JA, Alfonso A, Vieytes MR, Botana LM. Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines. Front Pharmacol. 2015;6:124. pmid:26136685
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref41] 41. Korsnes MS, Espenes A, Hetland DL, Hermansen LC. Paraptosis-like cell death induced by yessotoxin. Toxicol In Vitro. 2011;25(8):1764–70. pmid:21945047
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref42] 42. Fernandez-Araujo A, Alfonso A, Vieytes MR, Botana LM. Key role of phosphodiesterase 4A (PDE4A) in autophagy triggered by yessotoxin. Toxicology. 2015. Epub 2015/01/13.
View Article
Google Scholar

[156] View Article

[157] Google Scholar

[ref43] 43. Lai YW, Wang SW, Chang CH, Liu SC, Chen YJ, Chi CW, et al. Butein inhibits metastatic behavior in mouse melanoma cells through VEGF expression and translation-dependent signaling pathway regulation. BMC Complement Altern Med. 2015;15(1):445.
View Article
Google Scholar

[159] View Article

[160] Google Scholar

[ref44] 44. Becker JC, Houben R, Schrama D, Voigt H, Ugurel S, Reisfeld RA. Mouse models for melanoma: a personal perspective. Exp Dermatol. 2010;19(2):157–64. pmid:19849715
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref45] 45. Johnson DB, Sosman JA. Update on the targeted therapy of melanoma. Curr Treat Options Oncol. 2013;14(2):280–92. pmid:23420410
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref46] 46. Wang DY, Sosman JA, Johnson DB. Combination Immunotherapy: An Emerging Paradigm in Cancer Therapeutics. Oncology (Williston Park). 2015;29(12).
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref47] 47. Liu Y, Zhang G, Wang H, Liu S, Chen J, Zhao L, et al. Novel cyclic pentapeptide H-15 induces differentiation and inhibits proliferation in murine melanoma B16 cells. Oncol Lett. 2016;11(2):1251–5. pmid:26893727
View Article
PubMed/NCBI
Google Scholar

[173] View Article

[174] PubMed/NCBI

[175] Google Scholar

[ref48] 48. Vandercappellen J, Liekens S, Bronckaers A, Noppen S, Ronsse I, Dillen C, et al. The COOH-terminal peptide of platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly inhibits angiogenesis and suppresses B16 melanoma growth in vivo. Mol Cancer Res. 2010;8(3):322–34. pmid:20215425
View Article
PubMed/NCBI
Google Scholar

[177] View Article

[178] PubMed/NCBI

[179] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Reagents

Animals

Cells and cell lines

β-hexosaminidase release measurement

Determination of cellular viability and apoptosis/necrosis

B16F10 mouse model of subcutaneous melanoma

Glucose uptake measurement in B16F10 mouse model of subcutaneous melanoma

Statistical analysis

Results

Discussion

Author Contributions

References