Cinobufagin-induced DNA damage response activates G2/M checkpoint and apoptosis to cause selective cytotoxicity in cancer cells

Background Processed extracts from toad skin and parotoid gland have long been used to treat various illnesses including cancer in many Asian countries. Recent studies have uncovered a family of bufadienolides as the responsible pharmacological compounds, and the two major molecules, cinobufagin and bufalin, have been shown to possess robust antitumor activity; however, the underlying mechanisms remain poorly understood. Methods Intracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry, and DNA damage was analyzed by immunofluorescent staining and the alkaline comet assay. Cytotoxicity was measured by MTT as well as colony formation assays, and cell cycle and apoptosis were analyzed by flow cytometry. In addition, apoptosis was further characterized by TUNEL and mitochondrial membrane potential assays. Results Here we showed that sublethal doses of cinobufagin suppressed the viability of many cancer but not noncancerous cell lines. This tumor-selective cytotoxicity was preceded by a rapid, cancer-specific increase in cellular ROS and was significantly reduced by the ROS inhibitor N-acetyl cysteine (NAC), indicating oxidative stress as the primary source of cinobufagin-induced cancer cell toxicity. Sublethal cinobufagin-induced ROS overload resulted in oxidative DNA damage and intense replication stress in cancer cells, leading to strong DNA damage response (DDR) signaling. Subsequent phosphorylation of CDC25C and stabilization of p53 downstream of DDR resulted in activation of the G2/M checkpoint followed by induction of apoptosis. These data indicate that cinobufagin suppresses cancer cell viability via DDR-mediated G2 arrest and apoptosis. Conclusion As elevated oxidative pressure is shared by most cancer cells that renders them sensitive to further oxidative insult, these studies suggest that nontoxic doses of cinobufagin can be used to exploit a cancer vulnerability for induction of cancer-specific cytotoxicity.

A pore-forming protein drives macropinocytosis to facilitate toad water maintaining

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a secretory aerolysin family pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water intake and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporins, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

SAZONALIDADE DA PRODUTIVIDADE E PÓS-COLHEITA DE MELÃO 'PELE DE SAPO' SUBMETIDO À LÂMINAS DE IRRIGAÇÃO E DOSES DE BIOESTIMULANTE

SAZONALIDADE DA PRODUTIVIDADE E PÓS-COLHEITA DE MELÃO 'PELE DE SAPO' SUBMETIDO À LÂMINAS DE IRRIGAÇÃO E DOSES DE BIOESTIMULANTE DAÍSE SOUZA REIS LIMA1; WELSON LIMA SIMÕES2*; JOSÉ ALIÇANDRO BEZERRA DA SILVA3; MAGNO DO NASCIMENTO AMORIM4 E JUCICLÉIA SOARES DA SILVA5 * Autor for correspondence 1 Docente, Faculdade de Tecnologia e Ciências-UniFTC. Av. Clementino Coelho, 714 - Centro, CEP:56308-210, Petrolina – PE, Brasil. E-mail: [email protected] 2 Pesquisador – Embrapa Semiárido. BR 428, Km 152, CEP: 56302-970, Petrolina-PE, Brasil. E-mail: [email protected] 3 Docente, Universidade Federal do Vale do São Francisco, Av. Antônio C. Magalhães, 510 - Santo Antônio, Juazeiro - BA, CEP:48902-300, Juazeiro, BA, Brasil. E-mail: [email protected]; 4 Mestrando, Universidade Federal do Vale do São Francisco, Av. Antônio C. Magalhães, 510 - Santo Antônio, Juazeiro - BA, CEP:48902-300, Juazeiro, BA, Brasil. E-mail: [email protected] 5 Pós-doutoranda – Embrapa Semiárido. BR 428, Km 152, CEP: 56302-970, Petrolina-PE, Brasil. E-mail: [email protected] 1 RESUMO Buscando minimizar os efeitos sazonais nas plantas cultivadas em diferentes épocas, o presente estudo objetivou avaliar as respostas de produtividade e pós-colheita do melão ‘pele de sapo’ submetido à lâminas de irrigação e doses de bioestimulante em diferentes períodos no Semiárido brasileiro. As parcelas foram as lâminas de irrigação: 60; 80; 100; e 120% da evapotranspiração da cultura (ETc), as subparcelas foram cinco doses do bioestimulante Stimulate®: 0; 0,6; 1,2; 2,4 e 4,8 L ha-1, e as subsubparcelas foram dois períodos de cultivo: quente e frio. Foram avaliadas as variáveis produtividade total e comercial, peso médio do fruto comercial, sólidos solúveis totais (SST), pH, acidez total titulável (ATT) e firmeza de polpa (FP). A dose de 4,8 L ha-1 com a lâmina de 120% da ETc, no período quente, promoveram melhores condições de desenvolvimento e, consequentemente, um maior incremento na produtividade quando comparado com o período frio do semiárido brasileiro. A qualidade dos frutos foi influenciada pelo maior teor de SST no período quente e ATT no período frio, submetida à dose de 4,8 L ha-1 do Stimulate® e às lâminas abaixo de 100% da evapotranspiração da cultura. Palavras-chave: períodos, irrigação, Cucumis melo L., produtividade, qualidade do fruto. LIMA, D. S. R.; SIMÕES, W. L.; SILVA, J. A. B. da; AMORIM, M. DO N.; SILVA, J. S. da SEASONALITY OF PRODUCTION AND POST-HARVEST OF 'TOAD SKIN MELON' UNDER IRRIGATION DEPTHS AND DOSES OF BIOESTIMULANT 2 ABSTRACT Seeking to minimize seasonal effects on plants grown at different times, this study aimed to evaluate the productivity and post-harvest responses of melon ‘'toad skin' melon submitted to irrigation depths and doses of biostimulant in different periods in the Brazilian Semiarid. The plots were the irrigation depths: 60; 80; 100; and 120% of the culture evapotranspiration (ETc), the subplots were five doses of the Stimulate® biostimulant: 0; 0.6; 1.2; 2.4 and 4.8 L ha-1, and the sub-plots were two growing periods: hot and cold. The variables total and commercial productivity, the average weight of the commercial fruit, total soluble solids (SST), pH, total titratable acidity (ATT) and pulp firmness (FP) were evaluated. The dose of 4.8 L ha-1 with a depth of 120% of ETc, in the hot period, promoted better conditions of development and, consequently, a greater increase in productivity compared to the cold period of the Brazilian semiarid region. The quality of the fruits was influenced by the higher SST content in the hot period and ATT in the cold period, submitted to a dose of 4.8 L ha-1 of Stimulate® and to the depths below 100% of the crop evapotranspiration. Keywords: periods; irrigation, Cucumis melo L., productivity, fruit quality

Cinobufagin-Induced DNA Damage Response Activates G2/M Checkpoint and Apoptosis to Cause Selective Cytotoxicity in Cancer Cells

Background: Processed extracts from toad skin and parotoid gland have long been used to treat various illnesses including cancer in many Asian countries. Recent studies have uncovered a family of bufadienolides as the responsible pharmacological compounds, and the two major molecules, cinobufagin and bufalin, have been shown to possess robust antitumor activity; however, the underlying mechanisms remain poorly understood.Methods: Intracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry, and DNA damage was analyzed by immunofluorescent staining and the alkaline comet assay. Cytotoxicity was measured by MTT as well as colony formation assays, and cell cycle and apoptosis were analyzed by flow cytometry. In addition, apoptosis was further characterized by TUNEL and mitochondrial membrane poten­tial assays. Results: Here we showed that sublethal doses of cinobufagin suppressed the viability of many cancer but not noncancerous cell lines. This tumor-selective cytotoxicity was preceded by a rapid, cancer-specific increase in cellular ROS and was significantly reduced by the ROS inhibitor N-acetyl cysteine (NAC), indicating oxidative stress as the primary source of cinobufagin-induced cancer cell toxicity. Sublethal cinobufagin-induced ROS overload resulted in oxidative DNA damage and intense replication stress in cancer cells, leading to strong DNA damage response (DDR) signaling. Subsequent phosphorylation of CDC25C and stabilization of p53 downstream of DDR resulted in activation of the G2/M checkpoint followed by induction of apoptosis. These data indicate that cinobufagin suppresses cancer cell viability via DDR-mediated G2 arrest and apoptosis.Conclusion: As elevated oxidative pressure is shared by most cancer cells that renders them sensitive to further oxidative insult, these studies suggest that nontoxic doses of cinobufagin can be used to exploit a cancer vulnerability for induction of cancer-specific cytotoxicity.

Host defense or parasite cue: Skin secretions mediate interactions between amphibians and their parasites

Amphibian skin secretions (substances produced by the amphibian plus microbiota) plausibly act as a first line of defense against pathogen attack, but may also provide chemical cues for pathogens. To clarify the role of skin secretions in host-parasite interactions, we conducted experiments using cane toads (Rhinella marina) and their lungworms (Rhabdias pseudosphaerocephala) from the range-core and invasion-front of the introduced anurans’ range in Australia. Depending on the geographic area, toad skin secretions can reduce the longevity and infection success of parasite larvae, or attract lungworm larvae and enhance their infection success. These striking differences between the two regions were due both to differential responses of the larvae, and differential effects of the skin secretions. Our data suggest that skin secretions play an important role in host-parasite interactions in anurans, and that the arms race between a host and parasite can rapidly generate spatial variation in critical features of that interaction.

Melon cultivation irrigated with saline waters promote chemical alterations in an acrisol

The objective of the present study was to evaluate the salinization process and the changes in the chemical properties of an Acrisol cultivated with melon irrigated with water of different saline concentrations. The experiment was carried out in the field conditions using the experimental design of randomized blocks, arranged in split-plot. The plots were composed of salinity levels of irrigation water, expressed in terms of water electrical conductivity (ECw): 0.54; 1.48; 2.02 dS m-1; and the sub-plots for melon cultivars: Sancho and Medellín (Toad Skin), Mandacaru (Yellow), Nectar (Galia) and Sedna (Cantaloupe). Soil samples were collected at the beginning and end of the crop cycle to evaluate the changes in the physical-chemical properties of the soil. The electrical conductivity of saturation extract in the soil profile varied for each cultivar, observing high salinity values in soils with cultivars with lower water demand (Sedna and Nectar). The salinity of the soil was lower than the electrical conductivity of the irrigation water in the treatments with high values of ECw. The soil pH values showed little reaction in relation to the initial values. The exchangeable sodium percentage values were approximate twice the sodium adsorption ratio of the saturation stratum.

Heavy Metals Assessment in the Medjerda River Basin (Northeastern Algeria): A Preliminary Water Analysis and Toad Skin Biopsy

Our study attempted to monitor the quality of water in Medjarda basin (Northeastern Algeria) and to provide baseline information of heavy metals in the water as well as in a potential amphibian biosentinel, the spiny toad, Bufo spinosus. We measured pH, temperature, dissolved oxygen and biological oxygen demand of water and levels of heavy metals in toad skin using an atomic absorption flame spectrophotometer. Lead (Pb) concentration in water and in toad skin at all sites exceeded respectively 60 and 96 times the standard reference values. The heavy metal concentrations, in descending order, in water and in male toad skin were as follows: Pb > Fe > Cu > Zn and Fe > Pb > Zn > Cu respectively. This study highlights the ecological status of the surrounding areas upstream of the Medjarda basin as being a point source of heavy metal pollution. It is further stated that a non-invasive skin removal is an ethically sound technique to evaluate heavy metal accumulation in aquatic animals like toad, without euthanizing the specimens and making any loss to biodiversity of the species.

Hellebrigenin anti-pancreatic cancer effects based on apoptosis and autophage

Hellebrigenin is a natural product found in the toad skin secretions and plants of Urginea, including Hellebores and Kalanchoe genera. It has been shown to be active against Leishmania chagasi promastigotes and Trypanosoma cruzi trypomastigotes and also reported to play an anti-tumor effect on several cancer cell lines in vitro, including pancreatic cancer. This study is aimed to investigate the effects of Hellebrigenin on pancreatic carcinoma cells, SW1990 and BxPC-3 in vitro and its molecular mechanism involved in antitumor activities. Our results showed that Hellebrigenin effectively inhibited the proliferation of SW1990 and BxPC-3 cells in dose- and time-dependent manner. Flow cytometry results showed that Hellebrigenin induced the G0/G1 arrest in both of SW1990 and BxPC-3 cells and promoted cell early apoptosis and autophagy according to morphological observation. Immunofluorescence staining results further confirmed that cell apoptosis and autophagy also increased upon the Hellebrigenin treatment. Moreover, higher dose of Hellebrigenin further increased the cell apoptosis rate while decrease the mitochondrial membrane potential 24 h after treatment. The autophagy rate increased 48 h after treatment with significant difference (P < 0.05). Western blot analysis showed that the expression of caspase 3, 7, cleaved caspase 7, Atg 12, LC3 proteins were increased in SW1990 cell after treatment with Hellebrigenin. In addition, increasing expression of caspase 3, 7, 9, PARP, cleaved caspase 3, 7, 9, PARP, the sub basic protein of the PI3K family, Beclin-1, LC 3, Atg 3, 5, 12, 16 L were also observed after BxPC-3 cells treated with Hellebrigenin. In summary, this study reported for the first time that Hellebrigenin effectively induced autophagy and apoptosis especially the early apoptosis in SW1990 and BxPC-3 cells.