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Background: Moringa peregrina Forssk is a well-known plant in ethnomedicine due to its widespread uses in various diseases like cough, wound healing, rhinitis, fever, and detoxification. The plant seeds contain compounds that are cytotoxic to many cancer cells. During the therapeutic use of plants via the oral route, some compounds present in the plants may be cytotoxic to normal cell lines and red blood cells. Objective: This study was the first report of investigation of the cytotoxic profile on oral cancer, CAL 27, cell line, and hemolytic activities on human erythrocytes of Moringa peregrina seeds ethanolic extract (MPSE). Methods: MPSE was screened for its cytotoxic effect against oral cancer, CAL 27, cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT) assay. The toxicity of MPSE on human erythrocytes was determined by in vitro hemolytic assay. Results: MPSE showed significant anti-proliferative activity against oral cancer, CAL 27 cell line at lower concentrations with half maximal inhibitory concentration (IC50) value of 21.03 µg/mL. At 1,000 µg/ml of MPSE, the maximum hemolysis was found to be 14.3% which is within safer limit. Conclusions: This study revealed a potential anti-oral cancer of MPSE and provided a baseline for its potential use in oral cancer treatment with minimum hemolytic effect on human RBCs.
La Moringa peregrina Forssk es una planta muy conocida en etnomedicina debido a sus usos generalizados en diversas enfermedades como la tos, la cicatrización de heridas, la rinitis, la fiebre y la desintoxicación. Las semillas de la planta contienen compuestos citotóxicos para muchas células cancerosas. Durante el uso terapéutico de las plantas por vía oral, algunos compuestos presentes en ellas pueden ser citotóxicos para las líneas celulares normales y los glóbulos rojos. Objetivo: Este estudio fue el primer informe de investigación del perfil citotóxico sobre el cáncer oral, CAL 27, línea celular, y las actividades hemolíticas en eritrocitos humanos del extracto etanólico de semillas de Moringa peregrina (MPSE). Métodos: Se examinó el efecto citotóxico del MPSE contra la línea celular de cáncer oral CAL 27 mediante el ensayo con bromuro de 3-(4, 5-dimetiltiazol-2-il)-2, 5,-difeniltetrazolio (MTT). La toxicidad del MPSE sobre los eritrocitos humanos se determinó mediante un ensayo hemolítico in vitro. Resultados: MPSE mostró una actividad antiproliferativa significativa contra el cáncer oral, línea celular CAL 27 a concentraciones más bajas con un valor de concentración inhibitoria media máxima (IC50) de 21,03 µg/mL. A 1.000 µg/ml de MPSE, la hemólisis máxima fue del 14,3%, lo que está dentro del límite de seguridad. Conclusiones: Este estudio reveló un potencial anticancerígeno oral de MPSE y proporcionó una base para su uso potencial en el tratamiento del cáncer oral con un efecto hemolítico mínimo en los glóbulos rojos humanos.
Subject(s)
Humans , Moringa , Mouth Neoplasms , Cytotoxins , Erythrocytes , Medicine, TraditionalABSTRACT
Introducción. Los tumores neoplásicos se caracterizan por su invasividad y metástasis. Las células neoplásicas tienen heterogeneidad genética, por lo cual pueden desarrollar resistencia a los quimioterápicos. Por esta razón, las plantas continúan siendo una fuente importante de nuevos productos anticancerígenos. Objetivo. Evaluar la actividad citotóxica y antiproliferativa de un extracto rico en fucoidan de Lessonia trabeculata nativa (EFLt) sobre la línea celular de adenocarcinoma mamario murino, triple negativo 4T1. Métodos. La citotoxicidad y la IC50 se determinaron en monocapas de 4T1 empleando el reactivo MTT. Para demostrar la actividad antiproliferativa se aplicaron los métodos de cierre de herida y anticlonogénico utilizando las IC50 del EFLt y Dox (doxorubicina). El cierre de herida fue evaluado mediante barrido de tiempos discretos; el efecto anticlonogénico fue evaluado 7 días postratamiento mediante el conteo de colonias y se determinó la fracción de sobrevivencia. Adicionalmente, se evaluaron la citotoxicidad y actividad antiproliferativa combinando las IC50 de EFLt y Dox. El porcentaje de migración y conteo de colonias se realizó con el programa ImageJ. Resultados. La IC50 del EFLt (950 μg/mL) produjo 56% de citotoxicidad, 80,3% de inhibición de la migración celular, 68% de inhibición en la formación de colonias. La IC50 de Dox fue 0,5 μg/mL. Conclusiones. El EFLt ejerce citotoxicidad dependiente de la concentración y tiene efecto antiproliferativo sobre 4T1. Se requiere continuar los ensayos en modelos de mayor complejidad que permitan esclarecer el potencial antitumoral del EFLt.
Introduction. Neoplastic tumors are characterized by invasiveness and metastasis. Neoplastic cells are genetically heterogeneous and can develop resistance to chemotherapeutic agents. For this reason, plants continue to be an important source of new anticancer products. Objective. To determine the cytotoxic and antiproliferative activity of a fucoidan-rich extract of native Lessonia trabeculata (EFLt) on the tripe negative murine mammary adenocarcinoma cell line 4T1. Methods. Cytotoxicity and IC50 were determined in 4T1 monolayers using the MTT reagent. To demonstrate antiproliferative activity, "wound" closure and anticlonogenic methods were applied using the IC50 of EFLt and Doc (doxorubicin). "Wound" closure was evaluated by discrete times sweep to determine percentage inhibition of cell migration; the anticlonogenic effect was evaluated by colony counting 7 days after treatment and the survival fraction was determined. In addition, cytotoxicity and antiproliferative activity were evaluated by combining the IC50 of EFLt and Dox. Percent migration and colony counts were performed using ImageJ software. Results. The IC50 (950 μg/mL) of EFLt was 56% cytotoxicity, 80,3% inhibition of cell migration, 68% inhibition of colony formation.The ICof Dox was 0,5 μg/mL. Conclusions. EFLt exerts concentration-dependent cytotoxicity and antiproliferative effect on 4T1. Further studies in more complex models are needed to elucidate the anti-tumor potential of EFLt.
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Objective:To investigate the effect of transcription factor nuclear factor IB (NFIB) on cell proliferation and invasion in breast cancer.Methods:The lentivirus pLKO.1-shNFIB plasmid was constructed, packaged and infected with human estrogen receptor positive (ER + ) breast cancer cell line MCF-7 and triple-negative breast cancer (TNBC) cell line MDA-MB-231, respectively, NFIB was stably knocked down and verified by Western blot method; Cell count test (CCK-8) and clone formation test were used to investigate the effect of knockdown NFIB on the growth and proliferation of breast cancer cells; The transwell experiment and Western blot method were performed to detect the expression of epithelial mesenchymal transition protein markers. The effect of knockdown NFIB on the invasive ability of triple-negative breast cancer cells was explored; Kaplan-Meier survival was used to analyze web data (http: //kmplot.com/analysis/) to explore the effect of NFIB on the prognosis of ER + breast cancer and triple-negative breast cancer patients. Results:In MCF-7 and MDA-MB-231 breast cancer cells, knocking down NFIB inhibited cell growth and proliferation; In triple-negative breast cancer MDA-MB-231 cells, knocking down NFIB promoted the expression of interstitial marker fibronectin and promoted cell invasion; The lower the expression of NFIB, the worse the prognosis of triple negative breast cancer patients, while the expression of NFIB had no effect on the prognosis of ER + breast cancer patients. Conclusions:Knocking down NFIB inhibits the proliferation of MCF-7 cells, and the expression level of NFIB is not related to the prognosis of ER + breast cancer patients; Knocking down NFIB inhibits the proliferation of MDA-MB-231 cells but promotes their invasion; The low expression of NFIB is associated with the poor prognosis of triple-negative breast cancer patients.
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@#Objective To develop a method for monoclonal screening of CHO engineered cell lines using Verified In-situ Plate Seeding(VIPS). Methods Cell pool 1 stably transfected with target gene was inoculated by using monoclonal inoculation/imaging equipment(VIPS)in 96-well plates containing culture medium of different combinations(18 combinations,numbered 1～18)and volumes(100 and 200 μL/well). Monoclonal origin tracing pictures were taken,according to which,the monoclonal inoculation rate,clone formation rate and monoclonal proliferation rate were calculated and the image effects of monoclonal origin were evaluated. The applicability of this method was verified by cell pool 2,cell pool 3 and cell pool 4 stably transfected with target gene. Results The optimum monoclonal medium was Medium Ⅱ,and the volume of medium was 100 μL/well. Under the optimum process conditions,the monoclonal inoculation rate of Cell pool 1 was 80%,the clone formation rate was 83%,the proliferation rate was fast,and the monoclonal origin image was clear. While for Cell pool2,Cell pool 3 and Cell pool 4,the average rate of monoclonal inoculation was 78%,the average rate of clone formation was67%,the proliferation rate was slightly different and the image of cell division process was clear under the optimum process conditions. Conclusion In this study,the monoclonal screening method of CHO engineered cell lines developed using VIPS can improve the clone formation rate of CHO engineered cell lines and provide sufficient proof of monoclonal origin.
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@#Objective To develop a duplex digital PCR(dPCR) for evaluation of the stability of luciferase(Luc) gene in Luc2P reporter cell lines(CHO-K1,Hacat,HEK293 and UT7).Methods Genomic DNAs of Luc2P reporter cell lines were extracted,a duplex dPCR was developed to determine the copies of reference gene RPP30 and the target gene Luc,and the relative copy number of Luc(copies Luc/copy RPP30) was employed as the indicator for the stability evaluation of Luc gene;The developed method was verified for the specificity,precision,linearity,accuracy and durability,and analyzed for the applicability,according to the related requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Results All the original cells without reporter gene transfection were negative,while all the four reporter cell lines were positive,and the negative and positive regions in dPCR results were clearly distinguished;The relative standard deviation(RSD) of the eight repeated detections of the same genomic DNA sample and the six independent extractions of genomic DNA sample of the same cell were all less than 10%,and the linear fitting R~2 values were more than 0.99 for both Luc and RPP30.The spike recoveries of five groups of samples detected by the developed method were between 50% and 100%,and the results of chip-type dPCR and droplet-type dPCR were consistent.This method distinguished the relative copy number of Luc in different cell clones,and the results of detecting the relative copy number of Luc in three passages(P8,P12 and P31) were highly consistent.Conclusion The developed duplex dPCR method has good specificity,precision,linearity,accuracy,durability and applicability,and might be used to evaluate the stability of Luc gene in Luc2P reporter cell lines.
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Objective:To investigate whether interleukin(IL)-1β is involved in pyroptosis which leads to mouse islet β cell line βTC-6 cell damage, and to explore the role of JNK inhibitor SP600125 in inhibiting IL-1β induced βTC-6 cell pyroptosis.Methods:βTC-6 cell line and mouse islets were incubated with IL-1β for 48 h or intervened with both JNK inhibitor SP600125 and IL-1R antagonist IL-1Ra, then GSDMD expression and β cell pyroptosis morphology were detected by immunofluorescence staining of GSDMD and DAPI. The expression levels of Gsdmd, IL-1β and IL-18 mRNAs were detected by real time fluorescence PCR, and apoptosis was examined by Annexin-V/7-AAD staining combined with flow cytometry.Results:βTC-6 cell pyroptotic body was significantly increased in the IL-1β treated group compared with the control group, and the expressions of pyroptosis related genes Gsdmd, IL-1β, and IL-18 mRNA were significantly higher( P<0.05), and apoptosis was increased, suggesting that IL-1β effectively induced the βTC-6 cell pyroptosis, IL-1Ra prevented IL-1β induced βTC-6 cell pyroptosis. In the presence of JNK inhibitor SP600125, IL-1β treatment failed to induce the expressions of Gsdmd and IL-18 mRNA, markers of pyroptosis, and reduced the rate of apoptosis, indicating that SP600125 suppressed IL-1β induced βTC-6 cell pyroptosis. Conclusion:Pyroptosis is one of the mechanisms of βTC-6 cell impairment caused by IL-1β, and SP600125, a JNK inhibitor, can block the IL-1β induced pyroptosis pathway and has a potential role in inhibiting βTC-6 cell pyroptosis.
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Background Benzo[a]pyrene (BaP) has neurotoxicity, which can induce the loss of hippocampal neurons in humans and animals and lead to spatial learning and memory dysfunction, but its mechanism is still unclear. Objective To observe the ferroptosis of mouse hippocampal neuron HT22 cells induced by 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), an active metabolite of BaP, and to explore its potential mechanism, so as to provide a basis for the study of BaP neurotoxicity mechanism. Method Mouse hippocampal neuron HT22 cells were selected and divided into four groups: solvent control group and low, medium, and high concentration BPDE exposure groups (0.25, 0.50, and 0.75 μmol·L−1). Cell survival was detected by CCK8 method. Cell morphology and ultrastructure were observed under light and electron microscopes. The levels of reactive oxygen species (ROS) and Fe2+ were detected by fluorescence probe method. Iron, 4-hydroxynonenoic acid (4-HNE), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) levels were detected with commercial kits. The expression levels of acyl-CoA synthase long chain family member 4 (ACSL4), cyclooxygenase 2 (COX2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were detected by Western blotting. After interventions with ferroptosis inhibitors 20 μmol·L−1 deferoxamine (DFO) and 10 μmol·L−1 ethyl 3-amino-4-cyclohexylaminobenzoate (Fer-1), the cell survival rate of each BPDE exposure group and the changes of the ferroptosis characteristic indicators and protein expression levels were observed. Results With the increase of BPDE concentration, the survival rate of HT22 cells decreased gradually, and the survival rate of each BPDE group was significantly lower than that of the solvent control group (P<0.01). Under light microscope, the number of cells in the high concentration BPDE group was significantly reduced, and atrophic cells and reduced synapses were recorded. Under electron microscope, the HT22 cells in the high concentration BPDE group showed mitochondrial shrinkage, decreased crista, and increased mitochondrial membrane density. Compared with the solvent control group, the levels of intracellular lipid ROS, Fe2+, 4-HNE, and MDA significantly increased in the high concentration group (P<0.01), the GSH and GSH-PX levels were significantly decreased (P<0.01), the protein expression levels of ASCL4 and COX2 were significantly increased (P<0.01), and the protein expression levels of SCL7A11 and GPX4 were significantly decreased (P<0.01). The ferroptosis inhibitors DFO and Fer-1 significantly reversed the cell survival rate (P<0.01), the ferroptosis characteristic indicators (ROS, Fe2+, 4-HNE, MDA, GSH, and GSH-PX levels) (P<0.01), and the expression levels of ferroptosis-related proteins (ACSL4, COX2, SLC7A11, and GPX4) (P<0.01) in the high concentration BPDE group. Conclusion BPDE can induce ferroptosis in mouse hippocampal neuron HT22 cells, which may be related to the inhibition of SLC7A11/GSH/GPX4 axis and the induction of iron metabolism disorder.
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Objective:To observe the effect of rhGLP-1 (7-36) on Akt/GSK3 signaling pathway in hepatocytes.Methods:Human HL7702 cell line was cultured to the logarithmic growth stage and divided into experimental group and blank control group. The cultures were incubated with 100nM medium containing rhglp-1 (7-36) and without rhglp-1 (7-36) for 90min. The levels of Akt, Glycogen synthase Kinase 3 (GSK3) and Glycogen synthase (GS) in the two groups were detected by Western Blot.Results:Compared with blank control group, the protein expression of p-Akt (Thr308) in experimental group (1.81±0.28) was significantly increased ( P=0.01), but the protein expression of Akt and p-Akt (Ser473) was not significantly changed. The protein expression levels of p-GSK3α (Ser21) (1.27±0.09) and p-GSK3β (Ser9) (1.24±0.09) in the experimental group were significantly increased ( P=0.003, 0.002), while the protein expression levels of GSK3α and GSK3β were not significantly changed. The protein expression level of p-GS (Ser641) (0.70±0.16) was decreased in the experimental group ( P=0.03), but the protein expression level of GS did not change significantly. Conclusion:Glp-1 can inhibit GSK3/GS signaling pathway, activate GS activity and promote glycogen synthesis.
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OBJECTIVE@#To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism.@*METHODS@#MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 μg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry.@*RESULTS@#The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 μg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS.@*CONCLUSION@#DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.
Subject(s)
Humans , K562 Cells , Patrinia , Methylene Chloride/pharmacology , Apoptosis , Cell Proliferation , Cell DifferentiationABSTRACT
This study aimed to evaluate in vitro the possible mechanisms underlying the estrogenic potential of benzalkonium chloride (BAC) as a disinfectant emerging contaminant. Effects of BAC at the environmentally-relevant concentrations on estrogen synthesis and estrogen receptor (ER) signaling were assessed using the H295R steroidogenesis assay and the MCF-7 proliferation assay, respectively. Results showed that exposure to BAC at concentrations of 1.0-1.5 mg/L for 48 h significantly increased estradiol production of H295R cells in a concentration-dependent manner. Transcription of steroidogenic genes 3β‐HSD2, 17β‐HSD1, 17β‐HSD4, and CYP19A were significantly enhanced by BAC. In ER-positive MCF-7 cells, exposure to 0.5-1.5 mg/L BAC for 48 h significantly promoted cell proliferation and increased the expressions of ERα and G-protein coupled estrogen receptor 1. Flow cytometry analysis showed that 0.5-1.5 mg/L BAC significantly decreased the percentage of cells in G0/G1 phase, increased the percentage in S phase, and BAC at concentrations of 1.0 and 1.5 mg/L increased the G2/M phase cells. Findings of the study suggested that BAC at environmentally-relevant concentrations might act as a xenoestrogen through its inhibitory effect on steroidogenesis and ER-mediated mechanism.
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Brucellosis, a neglected tropical disease of zoonotic nature, is caused by the genus Brucella, specifically by Brucella abortus and B. melitensis in cattle and humans, respectively. Arjunolic acid (AA) is a triterpenoid, isolated from Terminalia arjuna (Roxb.) Wight & Arn., a medicinally important plant used to treat various diseases in the Indian system of medicine. Here, we tried to evaluate AA for its antibacterial activity on Brucella and the in vitro cytotoxicity assay on human lung adenocarcinomic alveolar basal epithelial cell line (A549). Also, we assessed the synergistic effect of arjunolic acid and Tarenna asiatica (L.) Kuntze ex K.Schum. on B. melitensis. AA displayed a considerable antibacterial activity [zone of inhibition (9 mm) with a minimum inhibitory concentration of 30 ?g/mL] against B. melitensis. The rate of cell death for the cancer cells were at 100 ?g/mL concentration of AA was 82% which indicates that AA shows significant membrane disruption to cancer cells. The estimated IC50 of AA against the A549 cell line was 139.90 ?g/mL. The highest synergistic activity was exhibited forming a zone of inhibition measuring 10mm when arjunolic acid and AqE of T. asiatica was added in the concentration of 1:1, respectively.
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BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.
Subject(s)
Animals , Recombinant Proteins/biosynthesis , CHO Cells , Caspase 7/genetics , Cell Cycle Checkpoints , Recombinant Proteins/genetics , Cell Division , Cricetulus , Cricetinae , Gene Knockout TechniquesABSTRACT
The aim of the present study was to evaluate the in vitro antiproliferative activity of ethanolic extract of leaves and fruits Citrus paradisi plant on HepG-2 liver cell lines by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Hterazolium bromide) assay and to isolate and characterize the antiproliferative compounds by TLC (Thin layer chromatography) and FT-IR (Fourier transforms Infrared) spectroscopy. Qualitative phytochemical screening tests were performed to detect phytochemicals compounds from the crude extracts. Antioxidant activity of the plant extracts were characterized by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging method. The results showed that antioxidant activity using DPPH were found to be increased in a concentration dependent manner and decreased cell viability and cell growth inhibition in a dose dependent manner. The findings from this study indicated that fruit extract exhibited good antiproliferation and antioxidant potential. The seven functional groups of phytocompounds such as carboxylic acid, amine salt, aromatic compounds, cyclic alkene, aldehyde, fluoro compounds and alkene were detected by FT-IR which indicated that fruit extracts of Citrus paradisi possessed vast potential as a medicinal drug especially in liver cancer treatment.
O objetivo do presente estudo foi avaliar a atividade antiproliferativa in vitro do extrato etanólico de folhas e frutos da planta Citrus paradisi em linhagens de células hepáticas HepG-2 por MTT (3- (4, 5-dimetil-2-tiazolil) -2, Ensaio de brometo de 5-difenil-2H-terazólio) e isolar e caracterizar os compostos antiproliferativos por espectroscopia de TLC (cromatografia de camada fina) e FT-IR (infravermelho com transformadas de Fourier). Testes qualitativos de triagem fitoquímica foram realizados para detectar compostos fitoquímicos nos extratos brutos. A atividade antioxidante dos extratos vegetais foi caracterizada pelo método de eliminação de radicais livres DPPH (2,2-difenil-1-picrilhidrazil). Os resultados mostraram que a atividade antioxidante usando DPPH aumentou de uma maneira dependente da concentração e diminuiu a viabilidade celular e a inibição do crescimento celular de uma maneira dependente da dose. Os resultados deste estudo indicaram que o extrato de fruta exibiu bom potencial antiproliferação e antioxidante. Os sete grupos funcionais de fitocompostos, como ácido carboxílico, sal de amina, compostos aromáticos, alceno cíclico, aldeído, compostos de flúor e alceno, foram detectados por FT-IR, o que indicou que extratos de frutas de Citrus paradisi possuíam vasto potencial como medicamento, especialmente no tratamento de câncer do fígado.
Subject(s)
Humans , Citrus paradisi , Liver Neoplasms/drug therapy , Plant Extracts/pharmacology , Cell Line , Spectroscopy, Fourier Transform Infrared , Phytochemicals , AntioxidantsABSTRACT
To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.
Subject(s)
Humans , CRISPR-Cas Systems/genetics , Gene Editing , Gene Knockout Techniques , HCT116 Cells , /metabolismABSTRACT
Abstract The aim of the present study was to evaluate the in vitro antiproliferative activity of ethanolic extract of leaves and fruits Citrus paradisi plant on HepG-2 liver cell lines by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide) assay and to isolate and characterize the antiproliferative compounds by TLC (Thin layer chromatography) and FT-IR (Fourier transforms Infrared) spectroscopy. Qualitative phytochemical screening tests were performed to detect phytochemicals compounds from the crude extracts. Antioxidant activity of the plant extracts were characterized by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging method. The results showed that antioxidant activity using DPPH were found to be increased in a concentration dependent manner and decreased cell viability and cell growth inhibition in a dose dependent manner. The findings from this study indicated that fruit extract exhibited good antiproliferation and antioxidant potential. The seven functional groups of phytocompounds such as carboxylic acid, amine salt, aromatic compounds, cyclic alkene, aldehyde, fluoro compounds and alkene were detected by FT-IR which indicated that fruit extracts of Citrus paradisi possessed vast potential as a medicinal drug especially in liver cancer treatment.
Resumo O objetivo do presente estudo foi avaliar a atividade antiproliferativa in vitro do extrato etanólico de folhas e frutos da planta Citrus paradisi em linhagens de células hepáticas HepG-2 por MTT (3- (4, 5-dimetil-2-tiazolil) -2, Ensaio de brometo de 5-difenil-2H-terazólio) e isolar e caracterizar os compostos antiproliferativos por espectroscopia de TLC (cromatografia de camada fina) e FT-IR (infravermelho com transformadas de Fourier). Testes qualitativos de triagem fitoquímica foram realizados para detectar compostos fitoquímicos nos extratos brutos. A atividade antioxidante dos extratos vegetais foi caracterizada pelo método de eliminação de radicais livres DPPH (2,2-difenil-1-picrilhidrazil). Os resultados mostraram que a atividade antioxidante usando DPPH aumentou de uma maneira dependente da concentração e diminuiu a viabilidade celular e a inibição do crescimento celular de uma maneira dependente da dose. Os resultados deste estudo indicaram que o extrato de fruta exibiu bom potencial antiproliferação e antioxidante. Os sete grupos funcionais de fitocompostos, como ácido carboxílico, sal de amina, compostos aromáticos, alceno cíclico, aldeído, compostos de flúor e alceno, foram detectados por FT-IR, o que indicou que extratos de frutas de Citrus paradisi possuíam vasto potencial como medicamento, especialmente no tratamento de câncer do fígado.
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Objective:To construct human immortalized keratinocytes stably expressing human papillomavirus type 16 (HPV16) E6/E7 gene, and provide a cell model for studying mechanisms underlying HPV16 E6/E7-induced cell immortalization and malignant transformation.Methods:Primary human foreskin keratinocytes (HFKs) were isolated by sequential two-step enzymatic digestion. Cultured HFKs were stably transfected with a HPV16 E6/E7 gene-overexpressing lentiviral vector LV5-HPV16 E6/E7, and consecutively cultured for more than 30 passages. Then, immortalized keratinocytes were screened out and divided into 3 groups: (1) blank control group: second-passage primary HFKs; (2) experimental group: HFKs transfected with LV5-HPV16 E6/E7 at different passages, and the second-passage primary HFKs transfected with LV5-HPV16 E6/E7 were referred to as A0 cells, thereafter, the transfected HFKs were named according to their passage number, such as A1, A2, ... A30; (3) positive control group: the HPV16-positive cervical cancer cell line SiHa. Real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the mRNA expression of HPV16 E6/E7 and protein expression of HPV16 E6/E7 and CK14, respectively, in the blank control group, experimental group and positive control group. Cell counting kit-8 (CCK8) assay and Transwell insert invasion assay were conducted to assess the cellular proliferative and invasive activity. In vivo tumor formation experiment in nude mice was conducted to investigate the tumorigenicity of A30 cells in the experimental group and SiHa cells in the positive control group. Results:Primary HFKs were successfully isolated. After the primary HFKs were transfected with the recombinant plasmid LV5-HPV16 E6/E7, the blank control group showed no fluorescence in the cells, but showed senescent phenotypes after serial passages, while in the experimental group, the volume and morphology of A30 cells were similar to those of the primary HFKs with the proportion of fluorescence-positive cells being 100%. Compared with the blank control group, the experimental group showed significantly increased mRNA expression levels of HPV16 E6 and E7 in A1, A10, A20 and A30 cells (HPV16 E6: t = 7.12, 8.07, 6.53, 5.66, P < 0.001, < 0.001, = 0.001, = 0.005, respectively; HPV16 E7: t = 3.20, 4.29, 3.75, 4.22; P = 0.024, 0.008, 0.013, 0.014, respectively) . The protein expression of HPV16 E6/E7 was absent in the blank control group, but was observed in A30 and SiHa cells. CCK8 assay showed that the proliferative activity of A10, A20 and A30 cells was significantly higher than that of the blank control group ( t = 6.49, 7.55, 9.43, P = 0.003, 0.002, 0.001, respectively) , while there was no significant difference in the proliferative activity between A1 cells and the blank control group ( t = 2.40, P = 0.074) . Transwell insert invasion assay showed that A30 cells could not cross the basement membrane, but SiHa cells could pass through the basement membrane and were stained blue. Two months after the inoculation with A30 cells in the nude mice, no visible tumors were found, which was confirmed by a histological study. Subcutaneous tumors were formed in the nude mice after the inoculation with SiHa cells. Conclusion:Human immortalized keratinocytes were successfully established by lentivirus-mediated transfection with HPV16 E6/E7 gene, and can serve as an ideal cell model for HPV-related research.
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Objective:To establish a melanoma cell line derived from a Chinese female patient of Han nationality with metastatic melanoma, and to study its basic biological characteristics.Methods:Metastatic melanoma cells were isolated from axillary lymph nodes of a 17-year-old female patient with malignant melanoma, and subjected to culture in vitro to establish a cell line. Short tandem repeat (STR) genotyping was performed to compare the information about the cell line and its derived tissue, and to detect gene mutations; cell counting kit-8 (CCK8) assay was conducted to assess the cellular proliferative activity, and soft agar cloning assay to assess the anchorage-independent proliferation; karyotype analysis was performed to determine the number and structure of chromosomes; with the highly aggressive melanoma cell line A2058 and keratinocyte line HaCaT serving as controls, Transwell assay was conducted to detect differences in cell migratory and invasive abilities, cell immunofluorescence assay and Western blot analysis were performed to determine the protein expression of melanoma-specific markers HMB45, S100 and Melan-A; the tumorigenicity was evaluated in vivo in a tumor-bearing mouse model. Results:A melanoma cell line was successfully established and named ZJMM-45, which was cultured for more than 70 passages over a 1-year period, and showed a stable shape and proliferative activity. The cells were spindle-shaped or polygonal, and could produce melanin. STR matching analysis showed that the ZJMM-45 sample was 96% matched with the cryopreserved lymph node tissue of the patient, suggesting that they were from the same source. A tumor-related gene BRAF V600E mutation (c.1799T>A) was identified in the ZJMM-45 cell line; karyotype analysis revealed that ZJMM-45 cells had triploid chromosomes and abnormal structures. ZJMM-45 cells grew exponentially in vitro and reached a plateau-phase in growth on day 5; the cells grew clonally and formed colonies in agar, showing anchorage-independent and malignant proliferative activity. Cell immunofluorescence assay and Western blot analysis showed that both ZJMM-45 and A2058 cells expressed HMB45, S100 and Melan-A; Transwell assay revealed that the numbers of invasive and migratory ZJMM-45 cells (300 ± 14, 260 ± 14, respectively) were significantly higher than those of invasive and migratory A2058 cells (150 ± 6, 160 ± 19, t = 13.25, 11.76, respectively, both P < 0.001) . The tumor-bearing mouse experiment showed that all 5 nude mice developed tumors with an inner diameter of 1.0 cm after 4 weeks, and the tumors were histopathologically characterized by proliferating hyperchromatic melanoma cell nuclei and formation of small nests, which were similar to primary solid tumors. Conclusion:The metastatic melanoma cell line ZJMM-45 derived from a Chinese patient with melanoma was successfully constructed, which carried the BRAF V600E mutation and expressed melanoma-specific markers, and was characterized by rapid proliferation, invasion and metastasis in both in vivo and in vitro culture, as well as obvious tumorigenicity in the in vivo experiment.
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Objective:To determine the expression of silent information regulator 1 (Sirt1) , Sirt3 and hypoxia-inducible factor 1α (HIF-1α) in cutaneous squamous cell carcinoma (CSCC) tissues and cells, and to explore their role in the occurrence and development of CSCC.Methods:From January 2019 to December 2020, 30 lesional skin tissues were obtained from patients with histopathologically confirmed poorly-, moderately- or well-differentiated CSCC, and 30 normal skin tissues were obtained from patients with non-cancerous diseases in Department of Dermatology, General Hospital of Ningxia Medical University. A CSCC cell line A431 and a human keratinocyte cell line HaCaT were cultured. Immunohistochemical study, Western blot analysis and real-time quantitative PCR (RT-PCR) were performed to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in CSCC tissues of different grades of differentiation and normal skin tissues, cytochemical and immunofluorescence staining and RT-PCR were conducted to determine the protein and mRNA expression of Sirt1, Sirt3 and HIF-1α in A431 and HaCaT cells, respectively. Comparisons of measurement data among multiple groups were performed by using one-way analysis of variance, and comparisons between two groups by using t test. Results:Immunohistochemical study showed that the expression level of Sirt3 (expressed as the average optical density) was 100 ± 12.12, 117.72 ± 26.23, 127.32 ± 24.45, 132.71 ± 31.61 in the normal skin tissues and well-, moderately- and poorly-differentiated CSCC tissues respectively, and there was a significant difference among these groups ( F = 20.14, P < 0.001) ; the expression of Sirt1 and HIF-1α increased in turn from the normal skin tissues to the well-, moderately- and poorly-differentiated CSCC tissues, and significantly differred in these groups ( F = 174.50, 225.00, respectively, both P < 0.001) . As Western blot analysis revealed, the expression level of Sirt3 significantly differed among the normal skin tissues, well-, moderately- and poorly-differentiated CSCC tissues (expressed as relative gray value: 1.000 ± 0.132, 1.403 ± 0.411, 1.387 ± 0.393, 1.677 ± 0.683, respectively; F = 34.97, P < 0.001) , and so did the expression levels of Sirt1 and HIF-1α ( F = 69.29, 199.90, respectively, both P < 0.00l) , with a gradually increasing trend in their expression levels from the the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues. RT-PCR showed that the mRNA expression of Sirt3, Sirt1 and HIF-1α was sequentially increased from the normal skin tissues to well-, moderately- and poorly-differentiated CSCC tissues, and significant differences were observed among these groups ( F = 113.00, 174.50, 50.33, respectively, all P < 0.001) . The protein expression levels of Sirt3, Sirt1 and HIF-1α were significantly higher in the A431 cells than in the HaCaT cells ( t = 16.75, 18.34, 27.76, respectively, all P < 0.001) , and so were their mRNA expression levels ( t= 14.22, 9.62, 16.86, respectively, all P < 0.001) . Conclusion:Increased expression of Sirt3, Sirt1 and HIF-1α was observed in CSCC tissues and cells, which may promote the occurrence and development of CSCC.
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Objective:To explore the effect of miR-21 on cell proliferation, apoptosis, invasion and radiosensitivity of cervical cancer HeLa cells and unravel the underlying mechanism.Methods:RT-qPCR assay was used to detect the expression levels of miR-21 in cervical cancer tissues and adjacent non-tumor tissues, normal cervical epithelial cells (H8) and cervical cancer cell lines (HeLa, SiHa, ME180). HeLa cell line with inhibition of miR-21 or knockdown of RECK were constructed. CCK-8, Caspase3/7 live cell apoptosis detection, wound healing test, Transwell invasion, clone formation assay, Western blot and immunofluorescence were performed to detect cell viability, apoptosis, migration, invasion, radiosensitivity and related proteins. The dual luciferase assay verified whether miR-21 targeted RECK.Results:MiR-21 level in the cervical cancer tissues was significantly higher than that in its corresponding adjacent non-tumor tissues ( P<0.05). The expression levels of miR-21 in cervical cancer cell lines HeLa, SiHa and ME180 were significantly up-regulated compared with those in normal cervical epithelial cells H8(all P<0.05). MiR-21 knockdown significantly inhibited HeLa cell viability, promoted cell apoptosis, reduced radiation tolerance, down-regulated the expression of Cyclin D 1,Bcl-2, MMP-2 and MMP-9, and up-regulated the expression P21 and Bax proteins (all P<0.05). miR-21 targeted the 3’-UTR of RECK mRNA and negatively regulated the expression of RECK. Silencing RECK reversed the effects of miR-21 knockdown on HeLa cell apoptosis, migration, invasion and radiosensitivity. Conclusions:Inhibiting the expression of miR-21 significantly decreases cell viability, induces cell apoptosis, weakens cell migration and invasion capabilities, and enhances the radiosensitivity of HeLa cells. The potential mechanism is closely related to the targeted up-regulation of RECK.
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Objective:To observe the effect of hyperthermia combined with paclitaxel on the proliferation, apoptosis and cycle of human tongue squamous cell carcinoma cell line CAL-27, and to explore the underlying mechanism.Methods:The working concentration of paclitaxel was determined by CCK-8 assay, and the cultured CAL-27 cells were divided into the control, paclitaxel, 42℃ hyperthermia and combined treatment groups. The ability of cell proliferation was detected by colony formation assay, and the cell cycle and apoptosis were determined by flow cytometry. The expression levels of AKT, p-AKT, Bcl-2 and Bax proteins in each group were measured by Western blot.Results:Compared with the control group, the proliferation was significantly inhibited and the apoptosis of CAL-27 cells was significantly promoted in the combined treatment, hyperthermia and paclitaxel groups (all P<0.05), and the anti-proliferation and apoptosis-promoting effect in the combined treatment group was significantly better than those in the hyperthermia and paclitaxel groups (all P<0.05). Western blot showed that hyperthermia combined with paclitaxel could significantly up-regulate the expression level of Bax protein and significantly down-regulate the expression levels of P-AKT and Bcl-2 in CAL-27 cells (all P<0.05). Conclusions:Hyperthermia combined with paclitaxel can play a synergistic role in inhibiting proliferation and promoting apoptosis of tongue squamous cell carcinoma CAL-27 cells. The mechanism may be related to the inhibition of AKT activation and the activation of Bax/Bcl-2 apoptosis signaling pathway.