RESUMO
Deoxypodophyllotoxin synthase (DPS), a nonheme Fe(II)/2-oxoglutarate (2OG)-dependent oxygenase, is a key enzyme that is involved in the construction of the fused-ring system in (-)-podophyllotoxin biosynthesis by catalyzing the C-C coupling reaction. However, the mechanistic details of DPS-catalyzed ring formation remain unclear. Herein, our quantum mechanics/molecular mechanics (QM/MM) calculations reveal a novel mechanism that involves the recycling of CO2 (a product of decarboxylation of 2OG) to prevent the formation of hydroxylated byproducts. Our results show that CO2 can react with the FeIII-OH species to generate an unusual FeIII-bicarbonate species. In this way, hydroxylation is avoided by consuming the OH group. Then, the C-C coupling followed by desaturation yields the final product, deoxypodophyllotoxin. This work highlights the crucial role of the CO2 molecule, generated in the crevice between the iron active site and the substrate, in controlling the reaction selectivity.
Assuntos
Oxirredução , Podofilotoxina , Podofilotoxina/química , Podofilotoxina/metabolismo , Podofilotoxina/análogos & derivados , Biocatálise , Estrutura Molecular , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Teoria Quântica , Medicamentos de Ervas ChinesasRESUMO
BACKGROUND: The study of cardiotoxicity of drugs has become an important part of clinical safety evaluation of drugs. It is commonly known that podophyllotoxin (PPT) and its many derivatives and congeners are broad-spectrum pharmacologically active substances. Clinical cardiotoxicity of PPT and its derivatives has been raised, basic research on the mechanism of cardiotoxicity remains insufficient. PURPOSE: In present study, our group's innovative concept of toxicological evidence chain (TEC) was applied to reveal the cardiac toxicity mechanism of PPT by targeted metabolomics, TMT-based quantitative proteomics and western blot. METHODS: The injury phenotype evidence (IPE) acquired from the toxicity manifestations, such as weight and behavior observation of Sprague-Dawley rat. The damage to rat hearts were assessed through histopathological examination and myocardial enzymes levels, which were defined as Adverse Outcomes Evidence (AOE). The damage to rat hearts was assessed through histopathological examination and myocardial enzyme levels, which were defined as evidence of adverse outcomes.Overall measurements of targeted metabolomics based on energy metabolism and TMT-based quantitative proteomics were obtained after exposure to PPT to acquire the Toxic Event Evidence (TEE). The mechanism of cardiac toxicity was speculated based on the integrated analysis of targeted metabolomics and TMT-based quantitative proteomics, which was verified by western blot. RESULTS: The results indicated that exposure to PPT could result in significant elevation of myocardial enzymes and pathological alterations in rat hearts. In addition, we found that PPT caused disorders in cardiac energy metabolism, characterized by a decrease in energy metabolism fuels. TMT-based quantitative proteomics revealed that the PPAR (Peroxisome proliferators-activated receptor) signaling pathway needs further study. It is worth noting that PPT may suppress the expression of SIRT1, subsequently inhibiting AMPK, decreasing the expression of PGC-1α, PPARα and PPARγ. This results in disorders of glucose oxidation, glycolysis and ketone body metabolism. Additionally, the increase in the expression of p-IKK and p-IκBα, leads to the nuclear translocation of NF-κB p65 from the cytosol, thus triggering inflammation. CONCLUSION: This study comprehensively evaluated cardiac toxicity of PPT and initially revealed the mechanism of cardiotoxicity,suggesting that PPT induced disorders of energy metabolism and inflammation via SIRT1/PPAR/NF-κB axis, potentially contributing to cardiac injury.
Assuntos
NF-kappa B , Podofilotoxina , Sirtuína 1 , Animais , Masculino , Ratos , Cardiotoxicidade , Coração/efeitos dos fármacos , Traumatismos Cardíacos/induzido quimicamente , Traumatismos Cardíacos/metabolismo , Metabolômica , Miocárdio/metabolismo , Miocárdio/patologia , NF-kappa B/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Proteômica , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismoRESUMO
Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.
Assuntos
Células HaCaT , Queratinócitos , Simulação de Acoplamento Molecular , Podofilotoxina , Tubulina (Proteína) , Humanos , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Podofilotoxina/química , Tubulina (Proteína)/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Corantes Fluorescentes/química , Sítios de Ligação , Estresse do Retículo Endoplasmático/efeitos dos fármacosRESUMO
To study the pharmacokinetics of deoxypodophyllotoxin and its metabolites in non-small cell lung cancer (NSCLC) bearing mice.Using the established LC-MS/MS method for simultaneous determination of deoxypodophyllotoxin and its three main metabolites (M1, M2 and M7) in biological samples, the concentrations of deoxypodophyllotoxin and its metabolites in plasma, tumour and major tissues of tumour-bearing mice were investigated after 6.25 and 25 mg/kg intravenous administration of deoxypodophyllotoxin.The exposure results of drug concentration showed that after intravenous injection of 6.25 and 25 mg/kg of DPT into tumour-bearing mice, the AUC ratio of DPT in tumour tissue to DPT in plasma was 4.23 and 3.80, respectively. While, the AUC ratio of metabolite M2 in tumour tissue to M2 in plasma was 0.82 and 0.76, respectively.Deoxypodophyllotoxin had higher affinity with tumour tissues than plasma, while its metabolite M2 had less affinity with tumour tissues than deoxypodophyllotoxin, but the exposure level of M2 in plasma was higher than that of deoxypodophyllotoxin. Deoxypodophyllotoxin was widely distributed in tumour-bearing mice. After intravenous injection of 25 mg/kg deoxypodophyllotoxin, the concentration of deoxypodophyllotoxin in other tissues except liver and muscle was relatively high, especially in lung, fat and reproductive organs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Podofilotoxina , Animais , Podofilotoxina/farmacocinética , Podofilotoxina/metabolismo , Podofilotoxina/análogos & derivados , Camundongos , Distribuição Tecidual , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas em Tandem , Medicamentos de Ervas ChinesasRESUMO
When new antitumor therapy drugs are discovered, it is essential to address new target molecules from the point of view of chemical structure and to carry out efficient and systematic evaluation. In the case of natural products and derived compounds, it is of special importance to investigate chemomodulation to further explore antitumoral pharmacological activities. In this work, the compound podophyllic aldehyde, a cyclolignan derived from the chemomodulation of the natural product podophyllotoxin, has been evaluated for its viability, influence on the cell cycle, and effects on intracellular signaling. We used functional proteomics characterization for the evaluation. Compared with the FDA-approved drug etoposide (another podophyllotoxin derivative), we found interesting results regarding the cytotoxicity of podophyllic aldehyde. In addition, we were able to observe the effect of mitotic arrest in the treated cells. The use of podophyllic aldehyde resulted in increased cytotoxicity in solid tumor cell lines, compared to etoposide, and blocked the cycle more successfully than etoposide. High-throughput analysis of the deregulated proteins revealed a selective antimitotic mechanism of action of podophyllic aldehyde in the HT-29 cell line, in contrast with other solid and hematological tumor lines. Also, the apoptotic profile of podophyllic aldehyde was deciphered. The cell death mechanism is activated independently of the cell cycle profile. The results of these targeted analyses have also shown a significant response to the signaling of kinases, key proteins involved in signaling cascades for cell proliferation or metastasis. Thanks to this comprehensive analysis of podophyllic aldehyde, remarkable cytotoxic, antimitotic, and other antitumoral features have been discovered that will repurpose this compound for further chemical transformations and antitumoral analysis.
Assuntos
Ciclo Celular , Podofilotoxina , Proteômica , Humanos , Podofilotoxina/farmacologia , Podofilotoxina/análogos & derivados , Podofilotoxina/química , Proteômica/métodos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Células HT29 , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacosRESUMO
In order to explore novel natural product-based insecticidal agent, two important intermediates (2 and 3) and 4-acyloxy-2'-bromo-6'-chloropodophyllotoxin derivatives (4 a-f and 5 a-f) were designed and prepared, and their structures were confirmed by 1H-NMR, 13C NMR, HRMS, ESI-MS, optical rotation and melting point (mp). The stereochemical configuration of compound 4 b was unambiguously confirmed by single-crystal X-ray diffraction. Moreover, we evaluated the insecticidal activity of target compounds 4 a-f and 5 a-f against a serious agricultural pest of Mythimna separata by using the leaf-dipping method. Among all tested compounds, compounds 4 d, 5 d and 5 f exhibited stronger insecticidal activity with a final mortality rate exceeding 60 %. Especially compound 5 d exhibited the best insecticidal activity, with a final mortality rate of 74.1 %. It has been proven that introducing bromine or chlorine atoms at the C-2', C-2' and C-6' positions of the E ring of podophyllotoxin can produce more potent compounds. In addition, the configuration of the C-4 position is important for insecticidal activity, and 4ß-configuration is optimal. This will pave the way for further design, structural modification, and development of derivatives of podophyllotoxin as insecticidal agents.
Assuntos
Inseticidas , Mariposas , Podofilotoxina , Inseticidas/síntese química , Inseticidas/farmacologia , Inseticidas/química , Animais , Podofilotoxina/farmacologia , Podofilotoxina/química , Podofilotoxina/síntese química , Podofilotoxina/análogos & derivados , Mariposas/efeitos dos fármacos , Relação Estrutura-Atividade , Estrutura Molecular , Cristalografia por Raios X , Conformação MolecularRESUMO
BACKGROUND: Leptomeningeal metastasis (LM) of small cell lung cancer (SCLC) is a highly detrimental occurrence associated with severe neurological disorders, lacking effective treatment currently. Proteolysis-targeting chimeric molecules (PROTACs) may provide new therapeutic avenues for treatment of podophyllotoxin derivatives-resistant SCLC with LM, warranting further exploration. METHODS: The SCLC cell line H128 expressing luciferase were mutated by MNNG to generate H128-Mut cell line. After subcutaneous inoculation of H128-Mut into nude mice, H128-LM and H128-BPM (brain parenchymal metastasis) cell lines were primarily cultured from LM and BPM tissues individually, and employed to in vitro drug testing. The SCLC-LM mouse model was established by inoculating H128-LM into nude mice via carotid artery and subjected to in vivo drug testing. RNA-seq and immunoblotting were conducted to uncover the molecular targets for LM. RESULTS: The SCLC-LM mouse model was successfully established, confirmed by in vivo live imaging and histological examination. The upregulated genes included EZH2, SLC44A4, VEGFA, etc. in both BPM and LM cells, while SLC44A4 was particularly upregulated in LM cells. When combined with PROTAC EZH2 degrader-1, the drug sensitivity of cisplatin, etoposide (VP16), and teniposide (VM26) for H128-LM was significantly increased in vitro. The in vivo drug trials with SCLC-LM mouse model demonstrated that PROTAC EZH2 degrader-1 plus VM26 or cisplatin/ VP16 inhibited H128-LM tumour significantly compared to VM26 or cisplatin/ VP16 alone (P < 0.01). CONCLUSION: The SCLC-LM model effectively simulates the pathophysiological process of SCLC metastasis to the leptomeninges. PROTAC EZH2 degrader-1 overcomes chemoresistance in SCLC, suggesting its potential therapeutic value for SCLC LM.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste , Neoplasias Pulmonares , Camundongos Nus , Podofilotoxina , Carcinoma de Pequenas Células do Pulmão , Animais , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Camundongos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Podofilotoxina/farmacologia , Podofilotoxina/análogos & derivados , Podofilotoxina/uso terapêutico , Linhagem Celular Tumoral , Carcinomatose Meníngea/tratamento farmacológico , Carcinomatose Meníngea/secundário , Ensaios Antitumorais Modelo de Xenoenxerto , Proteólise/efeitos dos fármacosRESUMO
The nonheme iron dioxygenase deoxypodophyllotoxin synthase performs an oxidative ring-closure reaction as part of natural product synthesis in plants. How the enzyme enables the oxidative ring-closure reaction of (-)-yatein and avoids substrate hydroxylation remains unknown. To gain insight into the reaction mechanism and understand the details of the pathways leading to products and by-products we performed a comprehensive computational study. The work shows that substrate is bound tightly into the substrate binding pocket with the C7'-H bond closest to the iron(IV)-oxo species. The reaction proceeds through a radical mechanism starting with hydrogen atom abstraction from the C7'-H position followed by ring-closure and a final hydrogen transfer to form iron(II)-water and deoxypodophyllotoxin. Alternative mechanisms including substrate hydroxylation and an electron transfer pathway were explored but found to be higher in energy. The mechanism is guided by electrostatic perturbations of charged residues in the second-coordination sphere that prevent alternative pathways.
Assuntos
Medicamentos de Ervas Chinesas , Hidrogênio , Ferro , Podofilotoxina/análogos & derivados , Oxirredução , Ferro/química , Hidroxilação , Hidrogênio/química , Estresse OxidativoRESUMO
Deoxypodophyllotoxin (DPT), a naturally occurring flavonolignan, possesses several pharmacological properties, including anticancer property. However, the mechanisms underlying DPT mode of action in oral squamous cell carcinoma (OSCC) remain unknown. This study aimed to investigate the anticancer effects of DPT on OSCC and the underlying mechanisms. Results of the MTT assay revealed that DPT significantly reduced the cell viability in a time- and dose-dependent manner. Flow cytometry analysis revealed that DPT induces apoptosis in OSCC cells in a dose-dependent manner. Moreover, DPT enhanced the production of mitochondrial reactive oxygen species (ROS) in OSCC cells. Mechanistically, DPT induced apoptosis in OSCC cells by suppressing the PI3K/AKT signaling pathway while activating the p38 MAPK signaling to regulate the expression of apoptotic proteins. Treatment with SC79, an AKT activator, reversed the effects of DPT on AKT signaling in OSCC cells. Taken together, these results provide the basis for the use of DPT in combination with conventional chemotherapy for the treatment of oral cancer.
Assuntos
Carcinoma de Células Escamosas , Flavonolignanos , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Apoptose , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Medicamentos de Ervas Chinesas , Flavonolignanos/farmacologia , Flavonolignanos/uso terapêutico , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Podofilotoxina/análogos & derivados , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The reactive oxygen species (ROS) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway play critical roles in the pathogenesis of prostate cancer by modulating cell proliferation. Picropodophyllin (PPP), an inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), exerts significant antitumor effects via the PI3K/AKT signaling pathway. However, the effects of PPP on prostate cancer via ROS production and the PI3K/AKT signaling pathway remain elusive. Herein, we focused on examining the antitumor effects of PPP on DU145 and LNCaP human prostate cancer cells to determine the possible molecular mechanism. Our data indicated that the inhibitory effect of PPP on the proliferation of DU145 and LNCaP human prostate cancer cells was mediated by apoptosis induction and cell cycle blockade. Furthermore, PPP significantly influenced the expression of apoptosis-related, cell cycle, ROS production, and PI3K/AKT signaling proteins. These findings suggest that PPP can induce cell cycle arrest and apoptosis via the production of ROS and inhibition of PI3K/AKT signaling pathway, thereby suppressing the proliferation of prostate cancer cells.
Assuntos
Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-akt , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases/metabolismo , Podofilotoxina/análogos & derivados , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de OxigênioRESUMO
Lignans constitute an important group of polyphenols, which have been demonstrated to potently suppress cancer cell proliferation. Numerous in vitro and in vivo studies indicate that deoxypodophyllotoxin as a natural lignan possesses potent anticancer activities against various types of human cancer. The purpose of current review is to provide the reader with the latest findings in understanding the anticancer effects and molecular mechanisms of deoxypodophyllotoxin. This review comprehensively describes the influence of deoxypodophyllotoxin on signaling cascades and molecular targets implicated in cancer cell proliferation and invasion. A number of various signaling molecules and pathways, including apoptosis, necroptosis, cell cycle, angiogenesis, vascular disruption, ROS, MMPs, glycolysis, and microtubules as well as NF-κB, PI3K/Akt/mTOR, and MAPK cascades have been reported to be responsible for the anticancer activities of deoxypodophyllotoxin. The results of present review suggest that the cyclolignan deoxypodophyllotoxin can be developed as a novel and potent anticancer agent, especially as an alternative option for treatment of resistant tumors to chemotherapy.
Assuntos
Antineoplásicos , Medicamentos de Ervas Chinesas , Lignanas , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Proliferação de Células , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Lignanas/farmacologia , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Podofilotoxina/uso terapêuticoRESUMO
Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.
Assuntos
Berberidaceae/enzimologia , Medicamentos de Ervas Chinesas/química , Ligases/química , Proteínas de Plantas/química , Podofilotoxina/análogos & derivados , Oxirredução , Podofilotoxina/químicaRESUMO
BACKGROUND: Podophyllotoxin is a natural lignan which possesses anticancer and antiviral activities. Etoposide and teniposide are semisynthetic glycoside derivatives of podophyllotoxin and are increasingly used in cancer medicine. OBJECTIVE: The present work aimed to design and synthesize a series of 2, 4, 5-trideoxyhexopyranosides derivatives of 4'-demethylepipodophyllotoxin as novel anticancer agents. METHODS: A divergent de novo synthesis of 2, 4, 5-trideoxyhexopyranosides derivatives of 4'- demethylepipodophyllotoxin has been established via palladium-catalyzed glycosylation. The abilities of synthesized glycosides to inhibit the growth of A549, HepG2, SH-SY5Y, KB/VCR and HeLa cancer cells were investigated by MTT assay. Flow cytometric analysis of cell cycle with propidium iodide DNA staining was employed to observe the effect of compound 5b on cancer cell cycle. RESULTS: Twelve D and L monosaccharide derivatives 5a-5l have been efficiently synthesized in three steps from various pyranone building blocks employing de novo glycosylation strategy. Dmonosaccharide 5b showed the highest cytotoxicity on five cancer cell lines with the IC50 values ranging from 0.9 to 6.7 µM. It caused HepG2 cycle arrest at G2/M phase in a concentrationdependent manner. CONCLUSION: The present work leads to the development of novel 2, 4, 5-trideoxyhexopyranosides derivatives of 4'-demethylepipodophyllotoxin. The biological results suggest that the replacement of the glucosyl moiety of etoposide with 2, 4, 5-trideoxyhexopyranosyl is favorable to their cytotoxicity. D-monosaccharide 5b was observed to cause HepG2 cycle arrest at the G2/M phase in a concentration- dependent manner.
Assuntos
Antineoplásicos , Podofilotoxina , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Células HeLa , Humanos , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Relação Estrutura-AtividadeRESUMO
Insulin-like growth factor-1 (IGF-1) primarily increases the release of gamma-aminobutyric acid (GABA) in neurons; moreover, it is responsible for the promotion of longitudinal growth in children and adolescents. Therefore, in this study, we investigated whether exogenous GABA supplementation activates IGF-mediated growth performance. Zebrafish larvae treated with GABA at three days post fertilization (dpf) showed a significant increase in the total body length from 6 to 12 dpf through upregulation of growth-stimulating genes, including IGF-1, growth hormone-1 (GH-1), growth hormone receptor-1 (GHR-1), and cholecystokinin A (CCKA). In particular, at 9 dpf, GABA increased total body length from 3.60 ± 0.02 to 3.79 ± 0.03, 3.89 ± 0.02, and 3.92 ± 0.04 mm at concentrations of 6.25, 12.5, and 25 mM, and the effect of GABA at 25 mM was comparable to 4 mM ß-glycerophosphate (GP)-treated larvae (3.98 ± 0.02 mm). Additionally, the highest concentration of GABA (50 mM) -induced death in 50% zebrafish larvae at 12 dpf. GABA also enhanced IGF-1 expression and secretion in preosteoblast MC3T3-E1 cells, concomitant with high levels of the IGF-1 receptor gene (IGF-1R). In zebrafish larvae, the GABA-induced growth rate was remarkably decreased in the presence of an IGF-1R inhibitor, picropodophyllin (PPP), which indicates that GABA-induced IGF-1 enhances growth rate via IGF-1R. Furthermore, we investigated the effect of GABA receptors on growth performance along with IGF-1 activation. Inhibitors of GABAA and GABAB receptors, namely bicuculline and CGP 46381, respectively, considerably inhibited GABA-induced growth rate in zebrafish larvae accompanied by a marked decrease in the expression of growth-stimulating genes, including IGF-1, GH-1, GHR-1, and CCKA, but not with an inhibitor of GABAC receptor, TPMPA. Additionally, IGF-1 and IGF-1R expression was impaired in bicuculline and CGP 46381-treated MC3T3-E1 cells, but not in the cells treated with TPMPA. Furthermore, treatment with bicuculline and CGP 46381 significantly downregulated GABA-induced IGF-1 release in MC3T3-E1 cells. These data indicate that GABA stimulates IGF-1 release via GABAA and GABAB receptors and leads to growth promotion performance via IGF-1R.
Assuntos
Receptores de GABA/metabolismo , Somatomedinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Ácido gama-Aminobutírico/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicerofosfatos/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Camundongos , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptores da Somatotropina/metabolismo , Peixe-Zebra/metabolismoRESUMO
Picropodophyllotoxin (PPT), an epimer of podophyllotoxin, is derived from the roots of Podophyllum hexandrum and exerts various biological effects, including anti-proliferation activity. However, the effect of PPT on colorectal cancer cells and the associated cellular mechanisms have not been studied. In the present study, we explored the anticancer activity of PPT and its underlying mechanisms in HCT116 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell viability. Flow cytometry was used to evaluate cell cycle distribution, the induction of apoptosis, the level of reactive oxygen species (ROS), assess the mitochondrial membrane potential (Δψm), and multi-caspase activity. Western blot assays were performed to detect the expression of cell cycle regulatory proteins, apoptosis-related proteins, and p38 MAPK (mitogen-activated protein kinase). We found that PPT induced apoptosis, cell cycle arrest at the G1 phase, and ROS in the HCT116 cell line. In addition, PPT enhanced the phosphorylation of p38 MAPK, which regulates apoptosis and PPT-induced apoptosis. The phosphorylation of p38 MAPK was inhibited by an antioxidant agent (N-acetyl-L-cysteine, NAC) and a p38 inhibitor (SB203580). PPT induced depolarization of the mitochondrial inner membrane and caspase-dependent apoptosis, which was attenuated by exposure to Z-VAD-FMK. Overall, these data indicate that PPT induced G1 arrest and apoptosis via ROS generation and activation of the p38 MAPK signaling pathway.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Podofilotoxina/análogos & derivados , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/química , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Podofilotoxina/química , Podofilotoxina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Chronic myeloid leukemia (CML) is a hematologic malignancy originating from BCR-ABL oncogene-transformed hematopoietic stem cells (HSCs) known as leukemia stem cells (LSCs). Therefore, targeting LSCs is of primary importance to eradicate CML. The present study demonstrates that picropodophyllin (PPP) effectively induces apoptosis and inhibits colony formation in CML stem/progenitor cells as well as quiescent CML progenitors resistant to imatinib therapy, while sparing normal hematopoietic cells in vitro. Administration of PPP in vivo markedly diminishes CML stem/progenitor cells in a transgenic mouse model of CML by inhibition of cell proliferation and enhancement of apoptosis in LSK cells, and significantly improves survival of CML mice. Furthermore, PPP treatment preferentially leads to transcriptional activation of p53 in CML but not normal CD34+ cells, upregulation of p53 protein in LSCs-enriched Sca-1+ cells from CML mice, and increased phosphorylation of p53 and upregulation of Bax protein in Ku812 cells. These results suggest that the inhibitory effects of PPP on CML stem/progenitor cells are associated with selective activation of p53 pathway and propose that PPP is a potent agent that selectively targets CML LSCs, and may be of value in the CML therapy.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Podofilotoxina/análogos & derivados , Células-Tronco/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Antígenos CD34/biossíntese , Apoptose , Modelos Animais de Doenças , Sangue Fetal/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Técnicas In Vitro , Células K562 , Camundongos , Camundongos Transgênicos , Microtúbulos/metabolismo , Fosforilação , Podofilotoxina/química , Transdução de Sinais , Ativação TranscricionalRESUMO
Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside.
Assuntos
Linho/metabolismo , Lignanas/biossíntese , Raízes de Plantas/metabolismo , Acetatos/metabolismo , Aminoácidos/biossíntese , Ciclopentanos/metabolismo , Indenos , Oxilipinas/metabolismo , Podofilotoxina/análogos & derivados , Podofilotoxina/biossínteseRESUMO
BACKGROUND: The aryltetralin lignan (-)-podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (-)-podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (-)-podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes. RESULTS: We recently established a four-step multi-enzyme cascade to convert (+)-pinoresinol into (-)-matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (-)-matairesinol to (-)-deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (-)-epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time. CONCLUSIONS: Both, (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Podofilotoxina/análogos & derivados , Podofilotoxina/biossíntese , Biocatálise , Biotransformação , Medicamentos de Ervas Chinesas , Escherichia coli/genética , Lignanas/metabolismoRESUMO
In our previous study, it showed that P-3F, a podophyllotoxin derivative, causes the increased level of p53 expression by enhancing p53 stability, resulting from blockage of the Mdm2-p53 feedback loop via nucleolus-to-nucleoplasm translocation of Rps27a in human cervical cancer HeLa cell line. However, the mechanism of regulating Rps27a localization remains to be studied. In the current study, it has been demonstrated that the level of protein interacting with carboxyl terminus 1 (PICT1), originally identified as a tumor suppressor, was decreased in a concentration-dependent manner in response to P-3F, leading to inhibition of human cervical cancer cell lines proliferation. Also remarkably, reduction of serine phosphorylation of STMN1 at position 16 induced by P-3F was required in the downregulation of PICT1, in which p53 activity was likely to be directly involved. Note as well that, PICT1 also played an important role in p53 stability enhancement by inhibiting Mdm2-mediated p53 ubiquitination due to Rps27a translocation from the nucleolus to the nucleoplasm to interact with Mdm2 following treatment with P-3F. Collectively, these findings indicated that P-3F, a microtubule polymerization inhibitor, promotes the decreased level of PICT1 expression, which is critical for regulating the Rps27a-Mdm2-p53 pathway against cervical cancer.
Assuntos
Antineoplásicos/farmacologia , Podofilotoxina/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinas/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Podofilotoxina/análogos & derivados , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Induction of macroautophagy (hereafter termed autophagy) is a strategy to improve the outcome of antineoplastic therapies by facilitating the induction of immunogenic cancer cell death and the consequent immune recognition of malignant cells. We analyzed 65,000 distinct compounds by means of a phenotypic discovery platform for autophagy induction and identified the IGF1R (insulin like growth factor 1 receptor) inhibitor picropodophyllin (PPP) as a potent inducer of autophagic flux. We found that PPP acts on-target, as an inhibitor of the tyrosine kinase activity of IGF1R and enhances the release of adenosine triphosphate, ATP, from stressed and dying cancer cells in vitro, thereby improving the therapeutic efficacy of chemoimmunotherapy in cancer-bearing mice. This PPP effect was phenocopied by another IGF1R inhibitor, linsitinib. Moreover, in human triple-negative breast cancer, phosphorylation of IGF1R correlates with reduced autophagy, an unfavorable local immune profile and poor prognosis. In summary, IGF1R inhibition may constitute a novel strategy for the treatment of cancer in the context of chemoimmunotherapy.