RESUMO
Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa.
Assuntos
Acetatos , Venenos Elapídicos , Indóis , Cetoácidos , Necrose , Mordeduras de Serpentes , Animais , Mordeduras de Serpentes/tratamento farmacológico , Camundongos , Humanos , Acrilamidas/farmacologia , Fosfolipases A2/metabolismo , Naja , Elapidae , Queratinócitos/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Reposicionamento de MedicamentosRESUMO
BACKGROUND: Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions. RESULTS: We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive 'spitting' or predatory 'biting' events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom. CONCLUSIONS: Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts.
Assuntos
Venenos de Serpentes , Serpentes , Animais , Venenos de Serpentes/químicaRESUMO
Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.
Assuntos
Neoplasias da Mama , Peixe-Zebra , Humanos , Feminino , Animais , Camundongos , Projetos Piloto , XenoenxertosRESUMO
Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well-documented structural diversity associated with PKS-derived natural products, C-C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of C-C-branched analogues. These C-C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains of jadomycins incorporating 3- and 4-aminomethylbenzoic acids. Labeling studies utilizing [1-13C]-d-glucose provided mechanistic evidence that the C-C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Carbono/metabolismo , Fibroblastos/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Policetídeo Sintases/metabolismo , Streptomyces/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Carbono/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Policetídeo Sintases/química , Células VeroRESUMO
Jadomycins are natural products that kill drug-sensitive and multidrug-resistant (MDR) breast cancer cells. To date, the cytotoxic activity of jadomycins has never been tested in MDR breast cancer cells that are also triple negative. Additionally, there is only a rudimentary understanding of how jadomycins cause cancer cell death, which includes the induction of intracellular reactive oxygen species (ROS). We first created a paclitaxel-resistant, triple-negative breast cancer cell line [paclitaxel-resistant MDA-MB-231 breast cancer cells (231-TXL)] from drug-sensitive control MDA-MB-231 cells (231-CON). Using thiazolyl blue methyltetrazolium bromide cell viability-measuring assays, jadomycins B, S, and F were found to be equipotent in drug-sensitive 231-CON and MDR 231-TXL cells; and using ROS-detecting assays, these jadomycins were determined to increase ROS activity in both cell lines by up to 7.3-fold. Jadomycins caused DNA double-strand breaks in 231-CON and 231-TXL cells as measured by γH2AX Western blotting. Coincubation with the antioxidant N-acetyl cysteine or pro-oxidant auranofin did not affect jadomycin-mediated DNA damage. Jadomycins induced apoptosis in 231-CON and 231-TXL cells as measured by annexin V affinity assays, a process that was retained when ROS were inhibited. This indicated that jadomycins are capable of inducing MDA-MB-231 apoptotic cell death independently of ROS activity. Using quantitative polymerase chain reaction, Western blotting, and direct topoisomerase inhibition assays, it was determined that jadomycins inhibit type II topoisomerases and that jadomycins B and F selectively poison topoisomerase IIß We therefore propose novel mechanisms through which jadomycins induce breast cancer cell death independently of ROS activity, through inhibition or poisoning of type II topoisomerases and the induction of DNA damage and apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dano ao DNA/fisiologia , Relação Dose-Resposta a Droga , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Isoquinolinas/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Multidrug resistance remains a major obstacle in the effective treatment of metastatic breast cancer. One mechanism by which multidrug resistance is conferred is the decreased intracellular drug accumulation due to the upregulation of the ATP-binding cassette (ABC) transporters. We have previously demonstrated that jadomycins, polyketide-derived natural products produced by Streptomyces venezuelae ISP5230, inhibit the growth of the human breast ductal carcinoma cell lines T47D and MDA-MB-435. To expand our understanding of jadomycin pharmacology, the goal of the present study was to determine whether the function of ABC efflux transporters affects the anticancer activity of jadomycins to MCF7 breast cancer cells. Seven jadomycin analogs (DNV, B, L, SPhG, F, S, and T) effectively reduced the viability of MCF7 control and ABCB1-, ABCC1-, or ABCG2-overexpressing drug-resistant MCF7 breast cancer cells as measured by methyltetrazolium cell viability assays and lactate dehydrogenase cytotoxicity assays. The inhibition of ABCB1, ABCC1, or ABCG2 with verapamil, MK-571, or Ko-143, respectively, did not augment the cytotoxicity of jadomycins DNV, B, L, SPhG, F, S, or T in drug-resistant MCF7 cells. Furthermore, jadomycins B, L, SPhG, F, S, and T did not increase the intracellular accumulation of ABCB1, ABCC1, or ABCG2 fluorescent substrates in HEK-293 cells stably transfected with ABCB1, ABCC1, or ABCG2. We conclude that jadomycins B, L, SPhG, F, S, and T are effective agents in the eradication of MCF7 breast cancer cells grown in culture, and that their cytotoxicities are minimally affected by ABCB1, ABCC1, and ABCG2 efflux transporter function.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Policetídeos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Aurora Quinase B/antagonistas & inibidores , Neoplasias da Mama , Sobrevivência Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células MCF-7 , Policetídeos/químicaRESUMO
Snakebites affect about 1.8 million people annually. The current standard of care involves antibody-based antivenoms, which can be difficult to access and are generally not effective against local tissue injury, the primary cause of morbidity. Here, we used a pooled whole-genome CRISPR knockout screen to define human genes that, when targeted, modify cell responses to spitting cobra venoms. A large portion of modifying genes that conferred resistance to venom cytotoxicity was found to control proteoglycan biosynthesis, including EXT1, B4GALT7, EXT2, EXTL3, XYLT2, NDST1, and SLC35B2, which we validated independently. This finding suggested heparinoids as possible inhibitors. Heparinoids prevented venom cytotoxicity through binding to three-finger cytotoxins, and the US Food and Drug Administration-approved heparinoid tinzaparin was found to reduce tissue damage in mice when given via a medically relevant route and dose. Overall, our systematic molecular dissection of cobra venom cytotoxicity provides insight into how we can better treat cobra snakebite envenoming.
Assuntos
Venenos Elapídicos , Mordeduras de Serpentes , Animais , Humanos , Mordeduras de Serpentes/tratamento farmacológico , Camundongos , Antídotos/farmacologiaRESUMO
Morbidity from snakebite envenoming affects approximately 400,000 people annually. Tissue damage at the bite-site often leaves victims with catastrophic life-long injuries and is largely untreatable by current antivenoms. Repurposed small molecule drugs that inhibit specific snake venom toxins show considerable promise for tackling this neglected tropical disease. Using human skin cell assays as an initial model for snakebite-induced dermonecrosis, we show that the drugs 2,3-dimercapto-1-propanesulfonic acid (DMPS), marimastat, and varespladib, alone or in combination, inhibit the cytotoxicity of a broad range of medically important snake venoms. Thereafter, using preclinical mouse models of dermonecrosis, we demonstrate that the dual therapeutic combinations of DMPS or marimastat with varespladib significantly inhibit the dermonecrotic activity of geographically distinct and medically important snake venoms, even when the drug combinations are delivered one hour after envenoming. These findings strongly support the future translation of repurposed drug combinations as broad-spectrum therapeutics for preventing morbidity caused by snakebite.
Assuntos
Mordeduras de Serpentes , Camundongos , Humanos , Animais , Mordeduras de Serpentes/tratamento farmacológico , Venenos de Serpentes/toxicidade , Venenos de Serpentes/uso terapêutico , Combinação de MedicamentosRESUMO
Snakebite envenoming is a global public health issue that causes significant morbidity and mortality, particularly in low-income regions of the world. The clinical manifestations of envenomings vary depending on the snake's venom, with paralysis, haemorrhage, and necrosis being the most common and medically relevant effects. To assess the efficacy of antivenoms against dermonecrosis, a preclinical testing approach involves in vivo mouse models that mimic local tissue effects of cytotoxic snakebites in humans. However, current methods for assessing necrosis severity are time-consuming and susceptible to human error. To address this, we present the Venom Induced Dermonecrosis Analysis tooL (VIDAL), a machine-learning-guided image-based solution that can automatically identify dermonecrotic lesions in mice, adjust for lighting biases, scale the image, extract lesion area and discolouration, and calculate the severity of dermonecrosis. We also introduce a new unit, the dermonecrotic unit (DnU), to better capture the complexity of dermonecrosis severity. Our tool is comparable to the performance of state-of-the-art histopathological analysis, making it an accessible, accurate, and reproducible method for assessing dermonecrosis in mice. Given the urgent need to address the neglected tropical disease that is snakebite, high-throughput technologies such as VIDAL are crucial in developing and validating new and existing therapeutics for this debilitating disease.
Assuntos
Mordeduras de Serpentes , Peçonhas , Humanos , Camundongos , Animais , Mordeduras de Serpentes/terapia , Antivenenos/farmacologia , Saúde Global , NecroseRESUMO
Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS. The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro. Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro, whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa.
RESUMO
Snakebite envenoming was reintroduced as a Category A Neglected Tropical Disease by the World Health Organization in 2017. Since then, increased attention has been directed towards this affliction and towards the development of a deeper understanding of how snake venoms exert their toxic effects and how antivenoms can counter them. However, most of our in vivo generated knowledge stems from the use of animal models which do not always accurately reflect how the pathogenic effects of snake venoms manifest in humans. Moreover, animal experiments are associated with pain, distress, and eventually animal sacrifice due to the toxic nature of snake venoms. Related to this, the implementation of the 3Rs principle (Replacement, Reduction, and Refinement) in the use of experimental animals in snakebite envenoming research is recommended by the World Health Organization. Therefore, more humane experimental designs and new in vitro/ex vivo alternatives for experimental animals are sought after. Here, we report the use of an organotypic model of human skin to further elucidate the pathophysiology of the dermonecrotic effects caused by the venom of the black-necked spitting cobra, Naja nigricollis, in humans. The goal of this study is to expand the repertoire of available models that can be used to study the local tissue damages induced by cytotoxic venoms.
Assuntos
Mordeduras de Serpentes , Animais , Humanos , Mordeduras de Serpentes/complicações , Proteômica , Venenos Elapídicos/toxicidade , Antivenenos/farmacologia , Naja , Venenos de SerpentesRESUMO
Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.
Assuntos
Antivenenos , Mordeduras de Serpentes , Animais , Antivenenos/uso terapêutico , Fibrinogênio , Mamíferos , Camundongos , Serina Proteases , Mordeduras de Serpentes/terapia , Venenos de Serpentes/toxicidade , Serpentes , Venenos de Víboras/toxicidadeRESUMO
Antivenom is currently the first-choice treatment for snakebite envenoming. However, only a low proportion of antivenom immunoglobulins are specific to venom toxins, resulting in poor dose efficacy and potency. We sought to investigate whether linear venom epitopes displayed on virus like particles can stimulate an antibody response capable of recognising venom toxins from diverse medically important species. Bioinformatically-designed epitopes, corresponding to predicted conserved regions of group I phospholipase A2 and three finger toxins, were engineered for display on the surface of hepatitis B core antigen virus like particles and used to immunise female CD1 mice over a 14 weeks. Antibody responses to all venom epitope virus like particles were detectable by ELISA by the end of the immunisation period, although total antibody and epitope specific antibody titres were variable against the different epitope immunogens. Immunoblots using pooled sera demonstrated recognition of various venom components in a diverse panel of six elapid venoms, representing three continents and four genera. Insufficient antibody yields precluded a thorough assessment of the neutralising ability of the generated antibodies, however we were able to test polyclonal anti-PLA2 IgG from three animals against the PLA2 activity of Naja nigricollis venom, all of which showed no neutralising ability. This study demonstrates proof-of-principle that virus like particles engineered to display conserved toxin linear epitopes can elicit specific antibody responses in mice which are able to recognise a geographically broad range of elapid venoms.
Assuntos
Formação de Anticorpos , Toxinas Biológicas , Animais , Antivenenos , Venenos Elapídicos/genética , Epitopos , Feminino , Camundongos , Venenos de SerpentesRESUMO
BACKGROUND: Snakebite is a major public health concern in Eswatini, where treatment relies upon one antivenom-SAIMR Polyvalent. Although effective in treating snakebite, SAIMR Polyvalent is difficult to source outside its manufacturing country (South Africa) and is dauntingly expensive. We compared the preclinical venom-neutralising efficacy of two alternative antivenoms with that of SAIMR Polyvalent against the lethal and tissue-destructive effects of venoms from five species of medically important snakes using in vivo murine assays. The test antivenoms were 'Panafrican' manufactured by Instituto Clodomiro Picado and 'PANAF' manufactured by Premium Serums & Vaccines. PRINCIPAL FINDINGS: In vivo murine preclinical studies identified both test antivenoms were equally or more effective than SAIMR Polyvalent at neutralising lethal and tissue-destructive effects of Naja mossambica venom. Both test antivenoms were less effective than SAIMR Polyvalent at neutralising the lethal effects of Bitis arietans, Dendroaspis polylepis, Hemachatus haemachatus and Naja annulifera venoms, but similarly effective at neutralising tissue damage induced by B. arietans and H. haemachatus venoms. In vitro immunological assays identified that the titres and toxin-specificities of immunoglobulins (iGs) in the test antivenoms were comparable to that of SAIMR Polyvalent. Plasma clotting disturbances by H. haemachatus and N. mossambica were neutralised by the test antivenoms, whereas SAIMR Polyvalent failed to neutralise this bioactivity of N. mossambica venom. B. arietans SVMP activity was equally reduced by all three antivenoms, and H. haemachatus and N. mossambica PLA2 activities were neutralised by all three antivenoms. CONCLUSIONS: While both Panafrican and PANAF antivenoms exhibited promising preclinical efficacies, both were less poly-specifically effective than SAIMR Polyvalent in these murine assays. The efficacy of these antivenoms against the lethal and tissue-destructive effects of N. mossambica venom, the most common biting species in Eswatini, identify that Panafrican and PANAF antivenoms offer effective alternatives to SAIMR Polyvalent for the treatment of snakebite in Eswatini, and potentially for neighbouring countries.
Assuntos
Mordeduras de Serpentes , Viperidae , Animais , Antivenenos/farmacologia , Antivenenos/uso terapêutico , Essuatíni , Camundongos , Fosfolipases A2 , Mordeduras de Serpentes/tratamento farmacológicoRESUMO
Snakebite envenoming is responsible for as many as 138â¯000 deaths annually, making it the world's most lethal neglected tropical disease (NTD). There is an urgent need to improve snakebite treatment, which currently relies on outdated and poorly tolerated biologic antivenoms that are often weakly efficacious, must be given intravenously in a healthcare setting, and are expensive to those who need them the most. Herein we describe the challenges associated with the discovery and development of new snakebite treatments and detail the great potential of venom toxin-inhibiting small molecule drugs. We finish by highlighting successful enabling strategies applied to other NTDs that could be exploited to facilitate the development of next-generation small molecule-based snakebite treatments.
Assuntos
Mordeduras de Serpentes , Antivenenos , Atenção à Saúde , Descoberta de Drogas , Humanos , Doenças Negligenciadas/tratamento farmacológico , Mordeduras de Serpentes/tratamento farmacológicoRESUMO
Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Metilação de DNA , Decitabina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cannabinoids exhibit anti-inflammatory and antitumorigenic properties. Contrary to most cannabinoids present in the Cannabis plant, some, such as O-1602 and abnormal cannabidiol, have no or only little affinity to the CB1 or CB2 cannabinoid receptors and instead exert their effects through other receptors. Here, we investigated whether the synthetic regioisomers of cannabidiol, abnormal cannabidiol, and a closely related compound, O-1602, display antitumorigenic effects in cellular models of breast cancer and whether it could reduce tumorigenesis in vivo. Several studies have shown the effects of cannabinoids on chemotherapy-sensitive breast cancer cell lines, but less is known about the antitumorigenic effects of cannabinoids in chemotherapy-resistant cell lines. Paclitaxel-resistant MDA-MB-231 and MCF-7 breast cancer cell lines were used to study the effect of O-1602 and abnormal cannabidiol on viability, apoptosis, and migration. The effects of O-1602 and abnormal cannabidiol on cell viability were completely blocked by the combination of GPR55 and GPR18-specific siRNAs. Both O-1602 and abnormal cannabidiol decreased viability in paclitaxel-resistant breast cancer cells in a concentration-dependent manner through induction of apoptosis. The effect of these cannabinoids on tumor growth in vivo was studied in a zebrafish xenograft model. In this model, treatment with O-1602 and abnormal cannabidiol (2 µM) significantly reduced tumor growth. Our results suggest that atypical cannabinoids, like O-1602 and abnormal cannabidiol, exert antitumorigenic effects on paclitaxel-resistant breast cancer cells. Due to their lack of central sedation and psychoactive effects, these atypical cannabinoids could represent new leads for the development of additional anticancer treatments when resistance to conventional chemotherapy occurs during the treatment of breast and possibly other cancers.
RESUMO
Herein, we report the characterization and antimicrobial activity of a previously unreported jadomycin (1) obtained from a culture of S. venezuelae ISP5230 with L-ornithine (Orn). 1 arises from the rearrangement of a putative five-membered ring containing jadomycin incorporating Orn, whereby intramolecular attack of the E-ring carbonyl from the δ-NH2 group of the Orn side chain results in collapse of the oxazolone ring and formation of a stable six-membered lactam. This rearrangement produces a jadomycin with a 3a hemiaminal position that is susceptible to solvolysis. A structure-activity relationship is discussed based on the antimicrobial activity of 1 compared to previously reported jadomycins, providing evidence that the presence of a 3a hemiaminal enhances activity against Gram-positive bacteria. Additionally, assays to quantify reactive oxygen species (ROS) generation and cell viability were performed using a series of nine jadomycins. Compound 1 was found to produce the highest ROS activity and to possess the greatest cytotoxicity against MDA-MB-231 breast cancer cells.