RESUMO
The development of novel drug candidates is a current challenge in pharmacology where therapeutic benefits must exceed side effects. Toxicology testing is therefore a fundamental step in drug discovery research. Herein, we describe the first line of toxicology testing program, consisting in cell-based high-throughput screening assays, which have the advantage of being easy, rapid, cheap, and reproducible while providing quantitative information. We illustrate MTT and Crystal Violet assays, two common colorimetric tests able to assess both cytostatic and cytotoxic effects, respectively, of a drug candidate. MTT assay allows evaluation of cellular metabolic activity, by which cell viability can be inferred; Crystal Violet staining is directly correlated with attached viable cells, thus allowing direct assessment of cell survival and death. Therefore, combination of the two methodologies represents a useful tool for predicting drug sensitivity and efficacy, the first milestones in pre-clinical toxicology workflow.
Assuntos
Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Violeta Genciana , Ensaios de Triagem em Larga Escala , Sais de Tetrazólio , Testes de Toxicidade , Testes de Toxicidade/métodos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Avaliação Pré-Clínica de Medicamentos/métodos , Sais de Tetrazólio/química , Ensaios de Triagem em Larga Escala/métodos , Animais , Colorimetria/métodos , Tiazóis/toxicidadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. AIM OF THE STUDY: The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. MATERIALS AND METHODS: We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305 cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. RESULTS: Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. CONCLUSIONS: These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.
Assuntos
Sobrevivência Celular , Mitocôndrias , Neurônios , Fármacos Neuroprotetores , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Trifosfato de Adenosina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Óxido Nítrico/metabolismo , Extratos Vegetais/farmacologia , Relação Dose-Resposta a Droga , Superóxidos/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Melastoma dodecandrum Lour. (MD), a traditional Chinese medicine used by the She ethnic group, has been used to treat cerebral ischemia-reperfusion (CIR) injury due to its efficacy in promoting blood circulation and removing blood stasiss; however, the therapeutic effects and mechanisms of MD in treating CIR injury remain unclear. AIM: To investigate the protective effects of MD on CIR injury, in addition to its impact on oxidative stress, endoplasmic reticulum (ER) stress, and cell apoptosis. MATERIALS AND METHODS: The research was conducted using both cell experiments and animal experiments. The CCK-8 method, immunofluorescence staining, and flow cytometry were used to analyze the effects of MD-containing serum on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cell viability, reactive oxygen species (ROS) clearance, anti-inflammatory, neuroprotection and inhibition of apoptosis. Furthermore, 2,3,5-Triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, Nissl staining, and immunohistochemistry were used to detect infarct size, pathological changes, Nissl corpuscula and neuronal protein expression in middle cerebral artery occlusion (MCAO) rats. Polymerase chain reaction and Western Blotting were conducted in cell and animal experiments to detect the expression levels of ER stress-related genes and proteins. RESULTS: The MD extract enhanced the viability of PC12 cells under OGD/R modeling, reduced ROS and IL-6 levels, increased MBP levels, and inhibited cell apoptosis. Furthermore, MD improved the infarct area in MCAO rats, increased the number of Nissl bodies, and regulated neuronal protein levels including Microtubule-Associated Protein 2 (MAP-2), Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP), and Neurofilament 200 (NF200). Additionally, MD could regulate the expression levels of oxidative stress proteins malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Both cell and animal experiments demonstrated that MD could inhibit ER stress-related proteins (GRP78, ATF4, ATF6, CHOP) and reduce cell apoptosis. CONCLUSION: This study confirmed that the therapeutic mechanism of the MD extract on CIR injury was via the inhibition of oxidative stress and the ER stress pathway, in addition to the inhibition of apoptosis.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Fármacos Neuroprotetores , Estresse Oxidativo , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Ratos , Células PC12 , Masculino , Fármacos Neuroprotetores/farmacologia , Apoptose/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêuticoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Triptolide is a major bioactive and toxic ingredient isolated from the traditional Chinese herb Tripterygium wilfordii (T. wilfordii) Hook F. It exhibits potent antitumor, immunosuppressive, and anti-inflammatory biological activities; however, its clinical application is hindered by severe systemic toxicity. Two preparations of T. wilfordii, including T. wilfordii glycoside tablets and T. wilfordii tablets, containing triptolide, are commonly used in clinical practice. However, their adverse side effects, particularly hepatotoxicity, limit their safe use. Therefore, it is crucial to discover potent and specific detoxification medicines for triptolide. AIM OF THE STUDY: This study aimed to investigate the detoxification effects and potential mechanism of action of spironolactone on triptolide-induced hepatotoxicity to provide a potential detoxifying strategy for triptolide, thereby promoting the safe applications of T. wilfordii preparations in clinical settings. MATERIALS AND METHODS: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Nuclear fragmentation was visualized using 4',6-diamidino-2-phenylindole (DAPI) staining, and protein expression was analyzed by Western blotting. The inhibitory effect of spironolactone on triptolide-induced hepatotoxicity was evaluated by examining the effects of spironolactone on serum alanine aminotransferase and aspartate aminotransferase levels, as well as liver pathology in a mouse model of triptolide-induced acute hepatotoxicity. Furthermore, a survival assay was performed to investigate the effects of spironolactone on the survival rate of mice exposed to a lethal dose of triptolide. The effect of spironolactone on triptolide-induced global transcriptional repression was assessed through 5-ethynyl uridine staining. RESULTS: Triptolide treatment decreased the cell viability, increased the nuclear fragmentation and the cleaved caspase-3 levels in both hepatoma cells and hepatocytes. It also increased the alanine aminotransferase and aspartate aminotransferase levels, induced the hepatocyte swelling and necrosis, and led to seven deaths out of 11 mice. The above effects could be mitigated by pretreatment with spironolactone. Additionally, molecular mechanism exploration unveiled that spironolactone inhibited triptolide-induced DNA-directed RNA polymerase II subunit RPB1 degradation, consequently increased the fluorescence intensity of 5-ethynyl uridine staining for nascent RNA. CONCLUSIONS: This study shows that spironolactone exhibits a potent detoxification role against triptolide hepatotoxicity, through inhibition of RPB1 degradation induced by triptolide and, in turn, retardation of global transcriptional inhibition in affected cells. These findings suggest a potential detoxification strategy for triptolide that may contribute to the safe use of T. wilfordii preparations.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Diterpenos , Compostos de Epóxi , Fenantrenos , Espironolactona , Compostos de Epóxi/toxicidade , Fenantrenos/toxicidade , Fenantrenos/farmacologia , Diterpenos/farmacologia , Diterpenos/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/patologia , Camundongos , Espironolactona/farmacologia , Masculino , Humanos , Sobrevivência Celular/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Células Hep G2RESUMO
BACKGROUND: Cervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. METHODS: The CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. RESULTS: E5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10-8 M (45.36 µg/ml) to 6.71 × 10-8 M (26.66 µg/ml) and Ifosfamide from 12.52 × 10-5 M (32.7 µg/ml) to 8.206 × 10-5 M (21.43 µg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. CONCLUSION: The study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.
Assuntos
Apoptose , Papillomavirus Humano 16 , Ifosfamida , Oxaliplatina , RNA Interferente Pequeno , Neoplasias do Colo do Útero , Humanos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Oxaliplatina/farmacologia , Feminino , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Ifosfamida/farmacologia , Apoptose/efeitos dos fármacos , Papillomavirus Humano 16/genética , Infecções por Papillomavirus/tratamento farmacológico , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Sobrevivência Celular/efeitos dos fármacos , Proteínas Oncogênicas Virais/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. AIM OF THE STUDY: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. MATERIALS AND METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 µg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 µg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.
Assuntos
Boraginaceae , Extratos Vegetais , Cicatrização , Peixe-Zebra , Cicatrização/efeitos dos fármacos , Animais , Extratos Vegetais/farmacologia , Humanos , Boraginaceae/química , Bioensaio , Linhagem Celular , Queratinócitos/efeitos dos fármacos , África do Sul , Células HaCaT , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacosRESUMO
Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34-phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.
Assuntos
Sobrevivência Celular , Leucemia Mieloide Aguda , Células-Tronco Mesenquimais , Fenótipo , Microambiente Tumoral , Humanos , Leucemia Mieloide Aguda/patologia , Microambiente Tumoral/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura/métodos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Medula Óssea/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Células da Medula Óssea/citologia , Masculino , Diferenciação Celular/efeitos dos fármacos , FemininoRESUMO
Controlled release or controlled drug delivery comprises the set of techniques and approaches to improve bioavailability through improved safety and/or efficacy using a carrier material for the molecule of interest. The predictability and tunability of these carriers make them ideal for protection, localization, and sustained presentation of a wide range of therapeutics, including growth factors implicated in cell survival and regeneration. Here we provide a method for encapsulating epidermal growth factor in a degradable polymer matrix for delivery to the cornea. Additional notes are included to demonstrate the wide-ranging capabilities of such methods for other materials, therapeutic agents, and sites of action within the eye.
Assuntos
Sobrevivência Celular , Preparações de Ação Retardada , Sobrevivência Celular/efeitos dos fármacos , Humanos , Regeneração , Fator de Crescimento Epidérmico/metabolismo , Animais , Córnea/metabolismo , Córnea/citologia , Sistemas de Liberação de Medicamentos/métodos , Polímeros/química , Portadores de Fármacos/químicaRESUMO
PURPOSE: Napabucasin (NP) is a natural compound that can suppress cancer cell proliferation and cell cycle by inhibition of the signal transducer and activator of transcription 3 (STAT3) gene. We examined the effects of NP on the proliferation and invasion of neuroblastoma cells (SH-SY5Y). METHODS: Human neuroblastoma SH-SY5Y cell line was used in this study. NP was administered to groups at the doses of 0.3-1 µM. Cell viability was analyzed by MTT assay. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis assessed the expressions of interleukin (IL)-6 dependent Jak2/Stat3 signaling pathway. The MTT cell viability method was applied to determine the antagonistic-synergistic effects and inhibitory concentration (IC50) doses of doxorubicin (DX) and NP. RESULTS: It was determined that 0.3-1 µM doses of NP killed the cells almost completely after 48 hours, and also that Jak2/Stat3 expressions decreased dose-dependently via IL-6. At the protein level, NP and DX were found to reduce Jak2 and Stat3 levels. CONCLUSIONS: NP showed that it suppresses the proliferation of neuroblastoma cells. Due to its inhibitory effect on Jak2 and Stat3, it can be used to prevent invasion of SH-SY5Y cells. NP, which can inactivate Jak2/Stat3, can be used as a treatment agent by combining with DX in proliferation pathway in neuroblastoma.
Assuntos
Benzofuranos , Proliferação de Células , Sobrevivência Celular , Doxorrubicina , Janus Quinase 2 , Neuroblastoma , Fator de Transcrição STAT3 , Transdução de Sinais , Humanos , Janus Quinase 2/metabolismo , Janus Quinase 2/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Doxorrubicina/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Sobrevivência Celular/efeitos dos fármacos , Benzofuranos/farmacologia , Interleucina-6/metabolismo , Western Blotting , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , NaftoquinonasRESUMO
OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.
Assuntos
Sobrevivência Celular , Difosfonatos , Endossomos , Fibroblastos , Gengiva , Hidrazonas , Imidazóis , Ácido Zoledrônico , Ácido Zoledrônico/farmacologia , Humanos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Gengiva/citologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Hidrazonas/farmacologia , Células Cultivadas , Fatores de Tempo , Reação em Cadeia da Polimerase em Tempo Real , Conservadores da Densidade Óssea/farmacologia , Reprodutibilidade dos Testes , Microscopia Confocal , Relação Dose-Resposta a Droga , Pinocitose/efeitos dos fármacosRESUMO
Docetaxel (DTX) has become widely accepted as a first-line treatment for metastatic breast cancer; however, the frequent development of resistance provides challenges in treating the disease.C60 fullerene introduces a unique molecular form of carbon, exhibiting attractive chemical and physical properties. Our study aimed to develop dicarboxylic acid-derivatized C60 fullerenes as a novel DTX delivery carrier. This study investigated the potential of water-soluble fullerenes to deliver the anti-cancer drug DTX through a hydrophilic linker. The synthesis was carried out using the Prato reaction. The spectroscopic analysis confirmed the successful conjugation of DTX molecules over fullerenes. The particle size of nanoconjugate was reported to be 122.13 ± 1.63 nm with a conjugation efficiency of 76.7 ± 0.14%. The designed conjugate offers pH-dependent release with significantly less plasma pH, ensuring maximum release at the target site. In-vitro cell viability studies demonstrated the enhanced cytotoxic nature of the developed nanoconjugate compared to DTX. These synthesized nanoscaffolds were highly compatible with erythrocytes, indicating the safer intravenous route administration. Pharmacokinetic studies confirmed the higher bioavailability (~ 6 times) and decreased drug clearance from the system vis-à-vis plain drug. The histological studies reveal that nanoconjugate-treated tumour cells exhibit similar morphology to normal cells. Therefore, it was concluded that this developed formulation would be a valuable option for clinical use.
Assuntos
Antineoplásicos , Neoplasias da Mama , Ácidos Carboxílicos , Sobrevivência Celular , Docetaxel , Sistemas de Liberação de Medicamentos , Fulerenos , Fulerenos/química , Fulerenos/administração & dosagem , Docetaxel/administração & dosagem , Docetaxel/farmacocinética , Docetaxel/farmacologia , Docetaxel/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Humanos , Feminino , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Ácidos Carboxílicos/química , Tamanho da Partícula , Portadores de Fármacos/química , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Nanoconjugados/química , Ratos , Células MCF-7 , Disponibilidade BiológicaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic lung disease. While recent studies have suggested the potential efficacy of tyrosine kinase inhibitors in managing IPF, masitinib, a clinically used tyrosine kinase inhibitor, has not yet been investigated for its efficacy in fibrotic lung diseases. In a previous study on an in vitro neurodegenerative model, we demonstrated the synergistic antitoxic and antioxidant effects of masitinib combined with cromolyn sodium, an FDA-approved mast cell stabilizer. This study aims to investigate the anti-fibrotic and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro model of pulmonary fibrosis. Fibroblast cell cultures treated with bleomycin and/or hydrogen peroxide (H2O2) were subjected to masitinib and/or cromolyn sodium, followed by assessments of cell viability, morphological and apoptotic nuclear changes, triple-immunofluorescence labeling, and total oxidant/antioxidant capacities, besides ratio of Bax and Bcl-2 mRNA expressions as an indication of apoptosis. The combined treatment of masitinib and cromolyn sodium effectively prevented the fibroblast myofibroblast transition, a hallmark of fibrosis, and significantly reduced bleomycin / H2O2-induced apoptosis and oxidative stress. This study is the first to demonstrate the additive anti-fibrotic, cell-protective, and antioxidant effects of the masitinib-cromolyn sodium combination in an in vitro fibrosis model, suggesting its potential as an innovative therapeutic approach for pulmonary fibrosis. Combination therapy may be more advantageous in that both drugs could be administered in lower doses, exerting less side effects, and at the same time providing diverse mechanisms of action simultaneously.
Assuntos
Antioxidantes , Apoptose , Benzamidas , Bleomicina , Cromolina Sódica , Fibroblastos , Miofibroblastos , Estresse Oxidativo , Piperidinas , Piridinas , Tiazóis , Antioxidantes/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Benzamidas/farmacologia , Piridinas/farmacologia , Apoptose/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Piperidinas/farmacologia , Cromolina Sódica/farmacologia , Animais , Tiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Sinergismo Farmacológico , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/patologia , Células Cultivadas , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologiaRESUMO
This work aimed to fabricate a Cloisite 30B-incorporated carboxymethyl cellulose graft copolymer of acrylic acid and itaconic acid hydrogel (Hyd) via a free radical polymerization method for controlled release of Sunitinib malate anticancer drug. The synthesized samples were characterized by FTIR, XRD, TEM, and SEM-dot mapping analyses. The encapsulation efficiency of Hyd and Hyd/Cloisite 30B (6 wt%) was 81 and 93%, respectively, showing the effectiveness of Cloisite 30B in drug loading. An in vitro drug release study showed that drug release from all samples in a buffer solution with pH 7.4 was higher than in a buffer solution with pH 5.5. During 240 min, the cumulative drug release from Hyd/Cloisite 30B (94.97% at pH 7.4) is lower than Hyd (53.71% at pH 7.4). Also, drug-loaded Hyd/Cloisite 30B (6 wt%) demonstrated better antibacterial activity towards S. Aureus bacteria and E. Coli. High anticancer activity of Hyd/Cloisite 30B against MCF-7 human breast cancer cells was shown by the MTT assay, with a MCF-7 cell viability of 23.82 ± 1.23% after 72-hour incubation. Our results suggest that Hyd/Cloisite 30B could be used as a pH-controlled carrier to deliver anticancer Sunitinib malate.
Assuntos
Carboximetilcelulose Sódica , Portadores de Fármacos , Hidrogéis , Indóis , Nanocompostos , Pirróis , Succinatos , Sunitinibe , Sunitinibe/química , Sunitinibe/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Succinatos/química , Succinatos/farmacologia , Carboximetilcelulose Sódica/química , Hidrogéis/química , Indóis/química , Indóis/farmacologia , Nanocompostos/química , Pirróis/química , Pirróis/farmacologia , Portadores de Fármacos/química , Células MCF-7 , Antineoplásicos/farmacologia , Antineoplásicos/química , Resinas Acrílicas/química , Administração Oral , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/administração & dosagem , Liberação Controlada de Fármacos , Staphylococcus aureus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Tobacco smoking is the leading cause of preventable death and disease worldwide, with over 8 million annual deaths attributed to cigarette smoking. This study investigates the impact of cigarette smoke and heated tobacco products (HTPs) on microglial function, focusing on toxicological profiles, inflammatory responses, and oxidative stress using ISO standard and clinically relevant conditions of exposure. METHODS: We assessed cell viability, reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial function, unfolded protein response, and inflammation in human microglial cells (HMC3) exposed to cigarette smoke, HTP aerosol or nicotine. RESULTS: Our findings show that cigarette smoke significantly reduces microglial viability, increases ROS formation, induces lipid peroxidation, and reduces intracellular glutathione levels. Cigarette smoke also alters the expression of genes involved in mitochondrial dynamics and biogenesis, leading to mitochondrial dysfunction. Additionally, cigarette smoke impairs the unfolded protein response, activates the NF-κB pathway, and induces a pro-inflammatory state characterized by increased TNF and IL-18 expression. Furthermore, cigarette smoke causes DNA damage and decreases the expression of the aging marker Klotho ß. In contrast, HTP, exhibited a lesser degree of microglial toxicity, with reduced ROS production, lipid peroxidation, and mitochondrial dysfunction compared to conventional cigarettes. CONCLUSION: These results highlight the differential toxicological profile of cigarette smoke and HTP on microglial cells, suggesting a potential harm reduction strategy for neurodegenerative disease for smokers unwilling or unable to quit.
Assuntos
Sobrevivência Celular , Inflamação , Peroxidação de Lipídeos , Microglia , Mitocôndrias , Estresse Oxidativo , Espécies Reativas de Oxigênio , Fumaça , Produtos do Tabaco , Resposta a Proteínas não Dobradas , Estresse Oxidativo/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Inflamação/patologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Produtos do Tabaco/efeitos adversos , Fumaça/efeitos adversos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Linhagem Celular , Temperatura Alta , NF-kappa B/metabolismo , Nicotiana/efeitos adversos , Dano ao DNARESUMO
Aging is a risk factor for various human disorders, including cancer. Current literature advocates that the primary principles of aging depend on the endogenous stress-induced DNA damage caused by reactive oxygen species 50 Hz low-frequency magnetic field was suggested to induce DNA damage and chromosomal instability. NF-kB, activated by DNA damage, is upregulated in age-related cancers and inhibition of NF-kB results in aging-related delayed pathologies. Metformin (Met), an NF-kB inhibitor, significantly reduces both NF-kB activation and expression in aging and cancer. This in vitro study, therefore, was set out to assess the effects of 5mT MF in 50 Hz frequency and Met treatment on the viability and proliferation of aged mouse NIH/3T3 fibroblasts and expression of RELA/p65, matrix metalloproteinases MMP2 and MMP9, and E-cadherin (CDH1) genes. The trypan blue exclusion assay was used to determine cell viability and the BrdU incorporation assay to determine cell proliferation. The MMP-2/9 protein analysis was carried out by immunocytochemistry, NF-kB activity by ELISA and the expressions of targeted genes by qRT-PCR methods. Four doses of Met (500 uM, 1 mM, 2 mM and 10 mM) suppressed both the proliferation and viability of fibroblasts exposed to the MF in a dose-dependent pattern, and the peak inhibition was recorded at the 10 mM dose. Met reduced the expression of NF-kB, and MMP2/9, elevated CDH1 expression and suppressed NF-kB activity. These findings suggest that Met treatment suppresses the carcinogenic potential of 50 Hz MFs in aged mouse fibroblasts, possibly through modulation of NF-kB activation and epithelial-mesenchymal transition modulation.
Assuntos
Proliferação de Células , Sobrevivência Celular , Fibroblastos , Campos Magnéticos , Metformina , NF-kappa B , Animais , Metformina/farmacologia , Camundongos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Células NIH 3T3 , NF-kappa B/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Fator de Transcrição RelA/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Caderinas/metabolismo , Caderinas/genética , Senescência Celular/efeitos dos fármacosRESUMO
The gut microbiota is a very important factor in the state of health of an individual, its alteration implies a situation of "dysbiosis," which can be connected to functional gastrointestinal disorders and pathological conditions, such as Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS), Ulcerative Colitis (UC) and Crohn's Disease (CD), and Colorectal Cancer (CRC). In this work, we studied the effect of a food supplement called ENTERO-AD containing a mix of probiotics (Lactobacillus acidophilus LA1, L. reuteri LR92, Bifidobacterium breve Bbr8), Matricaria Chamomilla, and B group vitamins (B1, B2, B6) on intestinal inflammation. The in vitro model used for the study is the Caco-2 cell, a culture derived from human intestinal adenocarcinoma; the inflammatory condition was achieved with treatment with Lipopolysaccharide (LPS) and the association between Tumor necrosis factor α/Interferon γ (TNF-α/IFN-γ) [1,2]. The effect of ENTERO-AD was evaluated by cell viability, measures of Transepithelial Electrical Resistance (TEER), paracellular permeability, and immunofluorescence. Results of the study have shown that ENTERO-AD has a favorable effect on Caco-2 cells in inflammatory conditions. It improves the integrity of Occludin and Zonula Occludens-1 (ZO-1) proteins, leading to an improvement in terms of TEER values and a reduction of paracellular permeability. This evidence underlines the protective effect of ENTERO-AD and its components in intestinal inflammation.
Assuntos
Suplementos Nutricionais , Mucosa Intestinal , Extratos Vegetais , Probióticos , Humanos , Probióticos/farmacologia , Probióticos/administração & dosagem , Células CACO-2 , Extratos Vegetais/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Vitaminas/farmacologia , Vitaminas/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Inflamação/patologia , Doenças Inflamatórias IntestinaisRESUMO
BACKGROUND: Autophagy plays a crucial role in modulating the proliferation of cancer diseases. However, the application of Naringenin (Nar), a compound with potential benefits against these diseases, has been limited due to its poor solubility and bioavailability. OBJECTIVE: This study aimed to develop solid lipid nanoparticles (Nar-SLNs) loaded with Nar to enhance their therapeutic impact. METHODS: In vitro experiments using Rin-5F cells exposed to Nar and Nar-SLNs were carried out to investigate the protective effects of Nar and its nanoformulation against the pancreatic cancer cell line of Rin-5F. RESULTS: Treatment with Nar and Nar-SLN led to an increase in autophagic markers (Akt, LC3, Beclin1, and ATG genes) and a decrease in the level of miR-21. Both Nar and Nar-SLN treatments inhibited cell proliferation and reduced the expression of autophagic markers. Notably, Nar-SLNs exhibited greater efficacy compared to free Nar. CONCLUSION: These findings suggest that SLNs effectively enhance the cytotoxic impact of Nar, making Nar-SLNs a promising candidate for suppressing or preventing Rin-5F cell growth.
Assuntos
Autofagia , Proliferação de Células , Flavanonas , Nanopartículas , Flavanonas/farmacologia , Flavanonas/administração & dosagem , Flavanonas/química , Autofagia/efeitos dos fármacos , Nanopartículas/química , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , Ratos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Lipídeos/química , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Humanos , Portadores de Fármacos/química , LipossomosRESUMO
The biosynthesis of nanoparticles is a crucial research area aimed at developing innovative, cost-effective, and eco-friendly synthesis techniques for various applications. Herein, we synthesized copper oxide nanoparticles (CuNPs) using Couroupita guianensis flower extract via a simple green synthesis method. These green CuNPs demonstrate promising antimicrobial activity and anticancer activity against A549 nonsmall cell lung cancer (NSCLC) cells. We comprehensively characterized the CuNPs using UV spectrum, XRD, FTIR, SEM, and EDS analyses. The antibacterial and anticancerous performance is attributed to their spherical-like morphology, which enhances effective interaction with bacterial and cancer cells. Moreover, CuNPs proved effective in inactivating Escherichia coli, achieving 2%, 52%, and 99% inactivation at 0, 30, and 60 min, respectively, and Listeria monocytogenes, achieving 1%, 48%, and 98% inactivation at 0, 30, and 60 min, respectively, under visible light. Furthermore, the CuNPs exhibited significant anticancer activity against A549 NSCLC cells, achieving cell viability reductions of 10%, 30%, 50%, 70%, 83%, and 91% at concentrations of 25, 50, 100, 150, 200, and 250 µg/mL, respectively. The green synthesized CuNPs demonstrate their potential in biomedical applications.
Assuntos
Antibacterianos , Antineoplásicos , Cobre , Escherichia coli , Flores , Nanopartículas Metálicas , Testes de Sensibilidade Microbiana , Extratos Vegetais , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Humanos , Cobre/química , Cobre/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Nanopartículas Metálicas/química , Flores/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Escherichia coli/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sobrevivência Celular/efeitos dos fármacos , Células A549 , Listeria monocytogenes/efeitos dos fármacos , Luminescência , Relação Dose-Resposta a Droga , Química Verde , Proliferação de Células/efeitos dos fármacosRESUMO
B-cell acute lymphoblastic leukemia (B-ALL), a prevalent malignancy predominantly affecting children, poses challenges such as drug resistance and cytotoxicity despite available treatment methods. The persistence of these challenges underscores the necessity for innovative therapeutic approaches to enhance efficacy. Natural compounds derived from plants, recognized for their potential to inhibit cancer cell growth, have drawn attention. Trifolium pratense extract, known for its significant anticancer properties in previous studies, was the focus of this investigation. This experimental study aimed to explore the impact of T. pratense extract on apoptosis and autophagy in NALM-6 cells. The cells were exposed to varying concentrations of the extract at specific time intervals, with viability and metabolic activity assessed using Trypan blue exclusion and MTT assays. Flow cytometry was employed to evaluate apoptosis using Annexin V/PI staining and ROS production using DCFH-DA staining. Real-time PCR was used to quantify gene expression related to apoptosis, autophagy, and oxidative stress, with data analysis performed using GraphPad PRISM software. Trifolium pratense extract demonstrated the capacity to induce apoptosis, autophagy, and significantly increase ROS production in NALM-6 cells. These effects were facilitated by the upregulation of corresponding genes. The MTT assay revealed an IC50 of 231 µg/mL at 48 h, and Flow cytometry analysis showed a 51.8% increase in apoptosis in this cell line. Overall, this study emphasizes the effectiveness of T. pratense extract in inducing autophagy and apoptosis pathways in NALM-6 cells derived from B-cell acute lymphoblastic leukemia, suggesting its potential as a candidate for further investigation as a supplement in ALL treatment.
Assuntos
Apoptose , Autofagia , Extratos Vegetais , Trifolium , Trifolium/química , Humanos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Extratos Vegetais/farmacologia , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologiaRESUMO
Nanoscale research is gaining interest in the biomedical, engineering, and environmental fields. Current expensive traditional chemical methods for synthesizing nanoparticles (NPs) inevitably lead to the synthesis of NPs with potentially less or no toxic effects on living cells. To overcome these challenges, in this study, we use a simple, inexpensive, and less toxic one-pot green chemistry approach instead of a chemical method to synthesize alumina nanoparticles (AlNPs) from Carica papaya extract. Nano-alumina has been widely studied due to its remarkable biological and physiochemical properties at nanoscale. However, to date, its biomedical application is limited due to the lack of sufficient data on cytotoxicity in living cells. The physicochemical properties of nano-alumina were determined by FT-IR, DLS, SEM and HRTEM. The cytotoxic effects of the synthesized nano-alumina were studied in cell lines LT and VERO at concentrations of 10-480 µg/mL in vitro. The cell viability of nano-alumina was evaluated using the MTT assay and the AO /EB double staining technique. Our results based on DLS and HRTEM analyzes confirmed spherical AlNPs with a zeta potential and average particle size of - 25 to 5 mV and 52 nm, respectively. The nano-alumina tested showed low toxicity to both cell lines after 28- and 48-h exposure. Furthermore, cell viability statistically decreased with increasing incubation time and concentration of AlNPs up to 480 µg/mL (p < 0.001). However, a minimal increase in cytotoxicity was observed at threshold levels in the range of 120-480 µg/mL. The half-maximal inhibitory concentration (IC50) of AlNPs in the VERO and LT cell lines were 153.3, 252.0 µg/mL and 186.6, 395.3 µg/mL, respectively, after 24- and 48-h exposure to AlNPs. Thus, we conclude that the cytotoxic effect of AlNPs depends on the concentration, exposure time and cell type. The result suggests that the concentration used in this study may be useful for biomedical applications.