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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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Objective:To evaluate the effect of circLPAR3 on the radiosensitivity of esophageal cancer cells and investigate its mechanism.Methods:The cancer tissues and and adjacent tissues of 37 patients with esophageal cancer were collected, and esophageal cancer cell lines Eca-109, EC9706 and KYSE30 and esophageal epithelial cells HET-1A were cultured in vitro. The expression levels of circLPAR3 and miR-1238 in the tissues and cells were measured by RT-qPCR. Eca-109 cells were transfected with circLPAR3 siRNA and miR-1238 mimics or co-transfected with circLPAR3 siRNA and miR-1238 inhibitor. Cell cloning experiment was conducted to evaluate the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 on the radiosensitivity of Eca-109 cells. After Eca-109 cells that silenced circLPAR3, overexpressed miR-1238 or silenced both circLPAR3 and miR-1238 were exposed to 4 Gy irradiation, CCK-8 assay (A value), flow cytometry and Western blot were employed to assess the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 combined with 4 Gy irradiation on the proliferation and apoptosis of Eca-109 cells and the expression levels of CyclinD1, p21, Bcl-2 and Bax proteins. Dual luciferase reporter gene experiment and RNA pull down experiment were performed to verify the regulatory relationship between circLPAR3 and miR-1238. Results:Compared with adjacent tissues, the expression level of circLPAR3 was up-regulated in the esophageal cancer tissues ( P<0.05), while that of miR-1238 was down-regulated ( P<0.05). Compared with HET-1A cells, the expression levels of circLPAR3 were up-regulated in the esophageal cancer cell lines Eca-109, EC9706 and KYSE30(all P<0.05), whereas those of miR-1238 were down-regulated (all P<0.05). Silencing circLPAR3 or overexpressing miR-1238 reduced the survival fraction of Eca-109 cells (all P<0.05), and the sensitization ratio was 1.21 and 1.75, respectively. Silencing circLPAR3 or overexpressing miR-1238 decreased the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins (all P<0.05), while increased the apoptosis rate of Eca-109 cells and the expression levels of p21 and Bax proteins (all P<0.05). After silencing circLPAR3 or overexpressing miR-1238 combined with 4 Gy irradiation, the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins were decreased (all P<0.05), while Eca-109 cell apoptosis rate and the expression levels of p21 and Bax proteins were increased (all P<0.05). circLPAR3 targeted and negatively regulated the expression level of miR-1238 in Eca-109 cells. After silencing miR-1238 and circLPAR3 simultaneously, the survival fraction of Eca-109 cells was higher than that when only silencing circLPAR3, and the sensitization ratio was 0.59. Silencing miR-1238 reversed the effects of silencing circLPAR3 combined with 4 Gy irradiation on the proliferation and apoptosis of Eca-109 cells. Conclusion:circLPAR3 is highly expressed in esophageal cancer tissues and cell lines, and silencing the expression of circLPAR3 can inhibit the proliferation of esophageal cancer Eca-109 cells, promote their apoptosis, and enhance cell radiosensitivity by up-regulating miR-1238.
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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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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
Abstract The main aim of the study is to quantify the cytotoxic property of the Fucoidan extracted from the Turbinaria conoides using the MTT assay with the standard fucose. Fucoidan was extracted using the soaked water method and it was determined using the HPLC procedure the obtained Test sample Fucoidan extracted from the Turbinaria conoides and standard fucose was subjected to the cytotoxicity assay against the MCF7 Human breast cancer cell line, A549 lung cancer cell line, and L929 normal mouse fibroblast cell line. From the results it was found that the Test sample showed good IC50 value for MCF7 cell line then A549 with an increasing concentration 24 hours incubation at 37°C The IC50 for MCF7 was 115.21 µg/ml and A549 396.46µg/ml and the Fucoidan extract was checked for its cytotoxicity against the normal mouse fibroblast cell line L929, Fucoidan was found non-lethal to the L929 mouse fibroblast normal cell line. Standard fucose also gave a significant result towards MCF7 and against the L929. This indicates that the Fucoidan extracted from Tubinaria conoides shows better anticancer potential in it. Hence its application can be further extended in the pharmacological fields.
Subject(s)
In Vitro Techniques/instrumentation , Cytotoxins/adverse effects , MCF-7 Cells , A549 Cells , Breast Neoplasms/pathology , Cell Line , Chromatography, High Pressure Liquid/methods , Inhibitory Concentration 50 , Fibroblasts/classification , Fucose/analogs & derivatives , Lung Neoplasms/pathologyABSTRACT
Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.
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Breast cancer is the most common cancer in women. At present, the in vivo model and traditional cell culture are mainly used in breast cancer researches. However, as high as 90% clinical trials are failed for drugs explored by the above two methods, due to the inherent species differences between humans and animals, as well as the differences in the tissue structure between organs and cells. Therefore, organoid three-dimensional culture is emerging. As a new tumor research model, organoid, a three-dimensional cell complex with spatial structure, has broad application prospects, such as precision medicine, organ transplantation, establishment of refractory disease model, gene therapy and drug research and development. Therefore, organoid is considered as one of the ideal carriers for life science research in the future. Breast cancer, a heterogeneous disease with complex phenotypes, has a low survival rate. Breast cancer organoid can reproduce many key features of human breast cancer, thus, the construction of organoid biological library of breast cancer will provide a new platform for studying the occurrence, development, metastasis and drug resistance mechanism of breast cancer. In this review, we systematically introduce the culture conditions of organoids and their application in breast cancer related research, and the application prospect of organoids.
Subject(s)
Animals , Female , Humans , Breast Neoplasms , Cell Culture Techniques , Organoids , Precision Medicine , ResearchABSTRACT
Objective:To investigate the effect of circ-PRKDC on lung cancer cell proliferation, apoptosis and radiosensitivity and its molecular mechanism.Methods:Normal lung epithelial cells BEAS-2B and lung cancer cell lines NCI-H1299, NCI-H2170, NCI-H1975 were cultured. NCI-H1299 cells were divided into the si-NC, si-PRKDC, pcDNA-NC, pcDNA-PRKDC, miR-NC, miR-505-3p, anti-miR-NC, anti-miR-505-3p, si-PRKDC+ anti-miR-NC and si-PRKDC+ anti-miR-505-3p groups. RT-qPCR was used to detect the expression levels of circ-PRKDC and miR-505-3p. Western blot was employed to measure the protein expression. MTT was used to detect cell proliferation. Flow cytometry was utilized to detect cell apoptosis. Plate clone formation assay was conducted to detect the cell radiosensitivity. Dual luciferase reporter assay was performed to analyze the targeting relationship between circ-PRKDC and miR-505-3p.Results:Compared with normal lung epithelial cells BEAS-2B, the expression levels of circ-PRKDC in the lung cancer cell lines NCI-H1299, NCI-H2170 and NCI-H1975 were significantly up-regulated (3.65, 3.10, 2.67 vs. 1.00, all P<0.05), whereas those of miR-505-3p were significantly down-regulated (0.42, 0.50, 0.54 vs. 1.02, all P<0.05). After low expression of circ-PRKDC, the expression level of CyclinD1 was significantly down-regulated (0.42 vs. 0.81, P<0.05), whereas those of Cleaved-caspase-3(0.71 vs. 0.33, P<0.05) and γ-H 2AX (0.89 vs. 0.46, P<0.05) were significantly up-regulated, the cell A value was significantly decreased (0.413 vs. 0.839, P<0.05), cell apoptosis rate was significantly increased (20.35 vs. 6.21, P<0.05), cell survival fraction was significantly decreased ( P<0.05), and β-catenin expression was significantly down-regulated (0.35 vs. 0.73, P<0.05). After high expression of miR-505-3p, the expression level of CyclinD1 was significantly down-regulated (0.34 vs. 0.83, P<0.05), those of Cleaved-caspase-3(0.65 vs. 0.32, P<0.05) and γ-H 2AX (0.96 vs. 0.45, P<0.05) were significantly up-regulated, the cell A value was significantly decreased (0.386 vs. 0.851, P<0.05), the apoptosis rate was significantly increased (16.38 vs. 6.20, P<0.05), and the cell survival fraction was significantly decreased ( P<0.05). Compared with miR-NC, the luciferase activity of miR-505-3p group transfected with circ-PRKDC wild-type reporter plasmid was significantly decreased (0.44 vs. 1.00, P<0.05). Down-regulation of miR-505-3p could reverse the effect of low expression of circ-PRKDC on the proliferation, apoptosis, radiosensitivity and β-catenin expression of NCI-H1299 cells. Conclusion:Low expression of circ-PRKDC may inhibit lung cancer cell proliferation, promote cell apoptosis and enhance cell radiosensitivity by up-regulating miR-505-3p, which is probably associated with the Wnt/β-catenin signaling pathway.
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Objective:To evaluate the effect of Salvianolic acid B on the radiosensitivity of human non-small cell lung cancer cells and investigate its possible mechanism.Methods:Non-small cell lung cancer cells A549 and H1299 were cultured in vitro. The toxicity of Salvianolic acid B on non-small cell lung cancer cells was detected by MTT assay. The effect of Salvianolic acid B on the radiosensitivity was assessed by clone formation assay. Transwell chamber assay was used to evaluate the effect of Salvianolic acid B on the migration of tumor cells. Western blot was employed to detect the expression levels of OTUD7B, MMP-2, MMP-9, E-cadherin, Akt and p-Akt regulated by Salvianolic acid B. Results:Salvianolic acid B (5 μmol/L) could inhibit the proliferation of A549 and H1299 cells. Clone formation assay showed that Salvianolic acid B increased the radiosensitivity of A549 and H1299 cells, with a radiosensitization ratio of 1.45 and 1.38, respectively. Transwell chamber assay indicated that the ability of cell migration was significantly inhibited by Salvianolic acid B ( P<0.05). Western blot revealed that the expression levels of OTUD7B in A549 and H1299 cells were induced by irradiation in a time-dependent manner. Salvianolic acid B could down-regulate the expression levels of MMP-2, MMP-9 and p-Akt, whereas up-regulate the expression level of E-cadherin by down-regulating the expression level of OTUD7B. Conclusions:Salvianolic acid B can enhance the radiosensitivity of A549 and H1299 cells. The possible mechanism is that Salvianolic acid B down-regulates the expression level of OTUD7B induced by irradiation and inhibits the epithelial-mesenchymal transition process of tumor cells.
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Objective:To examine the effect of FAM83D knockdown on proliferation, survival ability and invasion of human esophageal squamous cell carcinoma after X-ray radiation, and explore the mechanism.Methods:The expression of FAM83D, E-cadherin and vimentin in tumor tissues was detected in 69 cases of esophageal squamous cell cancer by using immunohistochemical method. The siRNA based on the sequences of the FAM83D mRNA were synthesized to transfect into the cultured ECA109 cells as FAM83D shRNA group. The effect of silencing FAM83D gene was evaluated to determine the protein levels of FAM83D in the human oesophageal squamous cell carcinoma ECA109 and KYSE30 cells using western blotting. MTS, clone formation, and Transwell assay were employed to examine the proliferation, survival ability and invasion of ECA109 and KYSE30 cells in vitro, respectively. We used flow cytometry assay to analyze distribution of cell apoptosis in different groups. Western blotting was used to examine the expression of cell metastasis-related molecules and apoptosis-related protein. Results:The strong expression rates of FAM83D, E-cadherin, and vimentin were 55%(38/69), 36%(25/69) and 61%(42/69) in the tumor tissues, respectively. FAM83D protein expression was significantly and negatively correlated with the expression of E-cadherin ( r=-0.350, P<0.01), and positively with the expression of vimentin ( r=0.470, P<0.01). Western blotting results demonstrated that silencing FAM83D gene significantly reduced the FAM83D protein expression ( P<0.01). MTS data demonstrated that FAM83D knockdown after irradiation significantly inhibited the proliferation of esophageal squamous cell carcinoma ECA109 and KYSE30 cells ( P<0.05). The data from the clone formation assay revealed that the radiosensitivity was increased after downragulation of FAM83D expression ( P<0.01). In addition, the invasive abilities of oesophageal carcinoma cells transfected with FAM83D shRNA after irradiation were significantly inhibited compared with those of the NC group ( P<0.01), followed by the downregulation of N-cadherin, vimentin, Snail, p-Akt and p-GSK-3β expression, and the upregulation of E-cadherin expression ( P<0.01). The apoptosis rate of tumor cells in FAM83D shRNA group after irradiation was markedly increased ( P<0.01), followed by a decrease of Bcl-2 and Mcl-1 expression and an increase of Cleaved caspase-3 expression ( P<0.01). Conclusions:FAM83D expressions was found to be closely related to the invasion and development of ESCC. Furthermore, siRNA interference technology inhibited the expression of FAM83D gene in oesophageal squamous cell carcinoma cells, reduced the proliferation, invasion of cells, induced cell apoptosis, and increased radiosensitivity, which may be associated with regulating the epithelial-mesenchymaltransition via Snail/Akt/GSK-3β signaling pathways.
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Objective:To clarify the role of classic Wnt signaling pathway in the radioresistance of esophageal cancer cells (ECC), and investigate the underlying mechanism, aiming to identify critical molecular targets for clinically enhancing the radiosensitivity of esophageal cancer.Methods:The radiosensitivity of four types of ECCs (EC9706, ECA109, KYSE70 and KYSE150) were assessed by colony formation assay. Western blot and RT-PCR were used to detect the activation of classical Wnt signaling pathway after irradiation. Classic Wnt signaling pathway activator (AZD2858) and inhibitor (XAV-939) were utilized to comprehensively evaluate the effect of classic Wnt signaling pathway on the radiosensitivity of ECCs. Cellular immunofluorescence staining was performed to detect the production and repair of DNA double-strand breaks (DSB), as well as the foci formation of DSB repair proteins after irradiation.Results:The results of colony formation assay showed that the radiosensitivity of four types of ECCs from high to low was EC9706, ECA109, KYSE70 and KYSE150. In KYSE150, a radioresistant cell type, the level of nuclear β-catenin and the transcription of c-Myc gene were significantly increased after irradiation (both P<0.05). However, in EC9706, a radiosensitive cell type, the level of nuclear β-catenin and c-Myc gene transcription were not affected by irradiation (both P>0.05). Moreover, EC9706 cells showed enhanced radioresistance in the presence of AZD2858( P<0.05), whereas XAV-939 treatment decreased the radioresistance in KYSE150 cells ( P<0.05). AZD2858 accelerated the DSB repair in EC9706 cells ( P<0.05), whereas XAV-939 delayed the DSB repair in KYSE150 cells ( P<0.05). Furthermore, the results of immunofluorescence staining showed that XAV-939 reduced the DSB repair capacity by inhibiting homologous recombination repair-related proteins (BRCA1 and RAD51) rather than non-homologous end junction repair-related proteins (Ku80 and XRCC4). Conclusions:The classic Wnt signaling pathway participates in the regulation of radiosensitivity in ECCs by regulating the homologous recombination repair of DSB after irradiation. Inhibition of the classic Wnt signaling pathway can counteract the radioresistance of ECCs and enhance the killing effect of irradiation on ECCs.
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Objection@#To investigate the effect of miR-32-5p on the radiosensitivity, migration and invasion of colorectal cancer cells and the underlying mechanism.@*Methods@#Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured. The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC, anti-miR-32-5p, pcDNA, pcDNA-TOB1, anti-miR-32-5p+ si-NC and anti-miR-32-5p+ si-TOB1, respectively). The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot. The radiosensitivity of the transfected cells was determined by colony formation assay. The migration and invasion ability of the transfected cells were detected by Transwell assay. Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.@*Results@#Compared with human colonic epithelial cells, the expression of miR-32-5p was significantly up-regulated, whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P<0.05). Compared with the anti-miR-NC, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group. Compared with the pcDNA group, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group. Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein. Compared with the anti-miR-32-5p+ si-NC group, the quantity of cell migration and invasion was significantly increased (both P<0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+ si-TOB1 group.@*Conclusions@#Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion. The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.
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Objective@#To explore the mechanism of the role of Salubrinal in regulating the radiation-induced apoptosis of oral cancer cells.@*Methods@#Radioresistant KBR cell line was constructed (4 Gy per fraction, every 7-10 d for 4 times). The radiosensitivity of oral cancer cells after Salubrinal pretreatment was measured by colony formation assay. The expression levels of NF-κB-HIF-1α signaling pathway and apoptosis biomarker cleaved PARP in oral cancer cells were measured by Western blot. The apoptosis rate was detected by Annexin V, PI staining and flow cytometry.@*Results@#Colony formation assay demonstrated that Salubrinal increased the radiosensitivity of oral cancer cells. The radiosensitization ratios of KB and KBR cells were 1.19 and 1.24. Western blot revealed that the activation of NF-κB-HIF-1α was time-dependent in the radiation-induced oral cancer cells, whereas Salubrinal inhibited the radiation-induced abnormal activation. In addition, Salubrinal increased the expression of apoptosis biomarker cleaved PARP and apoptosis index in radiation-induced oral cancer cells, whereas TNF-α, an activator of NF-κB, reversed the effect, suggesting that Salubrinal increased the apoptosis of radiation-induced oral cancer cells by suppressing the activation of NF-κB. Pretreatment of NF-κB inhibitor Bay11-7082 also increased the cell apoptosis. The expression levels of cleaved PARP of KB and KBR cell lines in the Bay11-7082+ IR group were 2.67±0.26 and 1.91±0.17, significantly higher compared with 2.1±0.16 and 1.44±0.15 in the IR group (both P<0.05).@*Conclusion@#Salubrinal can aggravate the apoptosis of radiation-induced oral cancer cells by inhibiting the radiation-induced activation of NF-κB, thereby regulating the radiosensitivity of oral cancer cells.
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Objection To investigate the effect of miR-32-5p on the radiosensitivity,migration and invasion of colorectal cancer cells and the underlying mechanism.Methods Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured.The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC,anti-miR-32-5p,pcDNA,pcDNA-TOB1,anti-miR-32-5p+si-NC and anti-miR-32-5p+si-TOB1,respectively).The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot.The radiosensitivity of the transfected cells was determined by colony formation assay.The migration and invasion ability of the transfected cells were detected by Transwell assay.Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.Results Compared with human colonic epithelial cells,the expression of miR-32-5p was significantly up-regulated,whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P＜0.05).Compared with the anti-miR-NC,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group.Compared with the pcDNA group,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group.Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein.Compared with the anti-miR-32-5p+si-NC group,the quantity of cell migration and invasion was significantly increased (both P＜0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+si-TOB1 group.Conclusions Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion.The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.
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Objective To explore the mechanism of the role of Salubrinal in regulating the radiation-induced apoptosis of oral cancer cells.Methods Radioresistant KBR cell line was constructed (4 Gy per fraction,every 7-10 d for 4 times).The radiosensitivity of oral cancer cells after Salubrinal pretreatment was measured by colony formation assay.The expression levels of NF-κB-HIF-1o signaling pathway and apoptosis biomarker cleaved PARP in oral cancer cells were measured by Western blot.The apoptosis rate was detected by Annexin V,PI staining and flow cytometry.Results Colony formation assay demonstrated that Salubrinal increased the radiosensitivity of oral cancer cells.The radiosensitization ratios of KB and KBR cells were 1.19 and 1.24.Western blot revealed that the activation of NF-κB-HIF-1α was time-dependent in the radiation-induced oral cancer cells,whereas Salubrinal inhibited the radiation-induced abnormal activation.In addition,Salubrinal increased the expression of apoptosis biomarker cleaved PARP and apoptosis index in radiation-induced oral cancer cells,whereas TNF-α,an activator of NF-κB,reversed the effect,suggesting that Salubrinal increased the apoptosis of radiation-induced oral cancer cells by suppressing the activation of NF-κB.Pretreatment of NF-κB inhibitor Bay1 1-7082 also increased the cell apoptosis.The expression levels of cleaved PARP of KB and KBR cell lines in the Bay11-7082+IR group were 2.67±0.26 and 1.91±0.17,significantly higher compared with 2.1±0.16 and 1.44±0.15 in the IR group (both P＜0.05).Conclusion Salubrinal can aggravate the apoptosis of radiation-induced oral cancer cells by inhibiting the radiation-induced activation of NF-κB,thereby regulating the radiosensitivity of oral cancer cells.
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Objective To evaluate the effect of ANO9 on the radiosensitivity of pancreatic cancer cell AsPC-1,aiming to provide new targets for clinical radiotherapy of pancreatic cancer.Methods Western blot was performed to detect the expression of ANO9 in pancreatic cancer cell lines (BxPC-3,PANC-1,AsPC-1)and normal pancreatic cell line (HPNE).The AsPC-1 cell line with stable silencing ANO9 was constructed by using lentivirus and validated by Western blot.MTT assay was adopted to detect the cell viability of AsPC-1 with stable silencing ANO9 after irradiation.Colony formation assay was conducted to evaluate the effect of silencing ANO9 upon the radiosensitivity of AsPC-1 cells.Western blot was performed to assess the effect of ANO9 silencing on the expression of EGFR/ERK signaling protein.Results The expression levels of ANO9 were significantly up-regulated in three pancreatic cancer cell lines compared with that in the normal pancreatic cell line HPNE (t =7.426,5.543,11.850,all P＜0.05).After silencing ANO9,the expression level of ANO9 protein was significantly down-regulated than that in the control group (t =9.670,P＜0.05).The AsPC-1 cells with stable silencing ANO9 were successfully constructed.The sensitivity of AsPC-1 cells to irradiation was significantly increased after silencing ANO9,and the sensitivity enhancement ratio was 1.566.The expression levels of EGFR/ERK signaling proteins (EGFR and p-ERK 1/2) were significantly downregulated after silencing ANO9 (t =7.949,13.160,both P＜ 0.05).Conclusions Silencing ANO9 can significantly increase the sensitivity of AsPC-1 cells to radiotherapy,which is probably associated with the inhibition of EGFR/ERK signaling transduction.ANO9 might be a new therapeutic target for preventing the progression of pancreatic cancer.
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Objective: To explore the expression of Ephrin receptor A7 (EphA7) in the patients with small cell lung cancer (SCLC) and its clinical significance. Methods: The expression of EphA7 mRNA in SCLC cell lines and tissues was analyzed by the Cancer Cell Line Encyclopedia (CCLE) and Oncomine databases, respectively. Seventy-three paraffin-embedded lung cancer specimens and six adjacent normal lung tissue samples from SCLC patients who underwent lobectomy or pneumonectomy resection in Tianjin Medical University Affiliated Cancer Hospital and Institution from January 2011 to December 2014 were collected. The expression of EphA7 protein was assessed by immunohistochemistry. The relationship between the expression of EphA7 protein and other clinicopathological factors was analyzed by x2 test. Kaplan-Meier survival curve and COX proportional hazard model were used to analyze the relationship between these clinicopathological parameters (including EphA7 expression) and the prognosis of SCLC patients. Results: The expression of EphA7 mRNA in SCLC cell lines was the highest among the 1 457 cell lines included in CCLE database. Three datasets of EphA7 mRNA expression in SCLC tissues were obtained from the Oncomine database. Compared with the normal lung tissues and non-small cell lung cancer, the expression level of EphA7 mRNA was relatively higher in SCLC tissues. The positive expression rate of EphA7 protein reached 72.6% (53/73) in the 73 patients with SCLC. The expression of EphA7 protein was significantly associated with lymph node metastasis and TNM stage (both P < 0.05). After adjusting other factors, it was found that the positive expression of EphA7 protein was an independent prognostic factor for the overall survival (OS) of SCLC patients [hazard ratio (HR) = 2.369, 95% confidence interval (CI): 1.075-5.219, P < 0.05], while TNM stage was an independent prognostic factor for both OS (HR = 2.273, 95% CI: 1.252-4.124, P < 0.05) and progression-free survival (PFS) (HR = 1.898, 95% CI: 1.088-3.312, P < 0.05) of SCLC patients, respectively. Conclusion: EphA7 mRNA and protein are highly expressed in SCLC tissues. The expression of EphA7 protein and TNM stage may be independent factors for the prognosis of SCLC patients.
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Objective@#To evaluate the effect of long-chain non-coding RNA TUG1 on the radiosensitivity of cervical cancer cells and explore its underlying mechanism.@*Methods@#The expression of TUG1 and miR-145 in cervical cancer cells XB1702 and normal endometrial stromal cells (ESCs) was detected by qRT-PCR. The transfected si-NC, transfected si-TUG1, transfected si-NC combined with irradiation, transfected si-TUG1 combined with irradiation, si-TUG1 and anti-miR-NC co-transfected and, si-TUG1 and anti-miR-145 co-transfected groups were established, which were transfected into XB1702 cells by liposome method. The survival fraction of each group was detected by colony formation assay. The cell apoptosis of each group was detected by flow cytometry. The fluorescence activity of each group was assessed by dual luciferase reporter gene assay.@*Results@#Compared with the normal ESCs, the expression of TUG1 was significantly up-regulated, whereas that of miR-145 was significantly down-regulated in the cervical cancer cells XB1702. Silencing TUG1 significantly increased the survival fraction of XB1702 cells, promoted cell apoptosis and enhanced the radiosensitivity of irradiation to XB1702 cells. TUG1 could target and regulate the expression of miR-145. Suppressing miR-145 reversed the silencing effect of TUG1 on inhibiting proliferation, accelerating apoptosis promotion and enhancing sensitization of XB1702 cells.@*Conclusions@#Silencing long-chain non-coding RNA TUG1 can enhance the radiosensitivity of cervical cancer cells. The mechanism may be related to targeting miR-145, which will provide a target for radiotherapy of cervical cancer.
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Objective:To define the anti-gastric cancer activity in vitro of petroleum ether fraction of Boehmeria nivea root and reveal the material basis of its efficacy, so as to lay the foundation for the development and utilization of B. nivea root. Method:Methyl thiazolyl tetraolium(MTT) method was used to evaluate the inhibitory rate and time-dose relationship of petroleum ether fraction of B. nivea root with different doses and delivery times on human gastric cancer HGC-27 cells. Flow cytometry was used to detect the change of cell apoptosis and cell cycle after petroleum ether fraction of B. nivea root acted on human gastric cancer HGC-27 cells. GC-MS was used to detect the components of petroleum ether fraction of B. nivea root. Result:Experiment data showed significant cell proliferation inhibition in an obvious time-dose-effect manner, with statistically significant differences (PB. nivea root. The effect of petroleum ether fraction of B. nivea root on human gastric cancer HGC-27 cells could induce apoptosis,which affects the normal changes of cell cycle. The percentage of cells was decreased significantly in G0/G1 phase,and that in S phase was significantly increased. GC-MS was used to identify 26 chemical constituents in petroleum ether of B. nivea root,including sitosterol and stigmasterol. Conclusion:Petroleum ether fraction of B. nivea root is the active anti-gastric cancer part,and its main effective component is sterol compounds. This lays the foundation for the rational application of B. nivea root in clinic and the further research in tis anti-tumor effect.
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@# Objective: To observe the effect of ursolic acid (UA) on autophagy and apoptosis of gastric cancer cell line MGC-803, and to explore the mechanism of UA-induced autophagy of MGC-803 cells based on PI3K/AKT/mTOR signaling pathway. Methods: Human gastric cancer cell line MGC-803 was cultured in vitro and divided into blank control group, UAintervention group and UA+3-MA group. The cell apoptosis in each group was detected by flow cytometry. Cell autophagy was detected by double fluorescence mRFPeGFP-LC3 plasmid transfection method. The mRNA expression levels of LC3B, BAX and Bcl-2 were detected by qPCR. The protein expression levels of PI3K type I, p-AKT, p-mTOR, ULK1, LC3B, BAX and Bcl-2 were detected by WB. Results: Flow cytometry showed that the cell apoptotic rate of UA intervention group was significantly higher than that of blank control group (P<0.05). Compared with UAintervention group, the apoptotic rate in UA+ 3-MAgroup was significantly reduced (P<0.05). The double fluorescence mRFP-eGFP-LC3 plasmid transfection method showed that the green and red fluorescent bright spots in UA intervention group increased significantly compared with the blank control group (P<0.05), and the green and red fluorescent bright spots in UA+3-MA group were significantly reduced compared with UA intervention group (P<0.05). Real-time quantitative PCR and WB method showed that compared with the blank control group, the mRNAand protein expressions of BAX and LC3B, and ULK1 protein were significantly increased in UA intervention group, while the mRNA and protein expressions of Bcl-2, and the protein expressions of PI3K, p-AKT and p-mTOR were significantly decreased in UA intervention group (all P<0.05); Compared with UA intervention group, mRNA and protein expressions of BAX and LC3B were significantly down-regulated and the mRNAand protein expressions of Bcl-2 were significantly up-regulated in UA+3-MA group (all P<0.05), while protein levels of PI3K, p-AKT, p-mTOR and ULK1 were not significantly changed in UA+3-MA group (P>0.05). Conclusion: UA can promote apoptosis of MGC-803 cells via inducing autophagy, which may be related to UA's involvement in regulating the expressions of PI3K/AKT/mTOR signaling pathway-related proteins.