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Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death worldwide. It is urgent to develop new drugs to improve the prognosis of ESCC patients. Here, we found benzydamine, a locally acting non-steroidal anti-inflammatory drug, had potent cytotoxic effect on ESCC cells. Benzydamine could suppress ESCC proliferation in vivo and in vitro. In terms of mechanism, CDK2 was identified as a target of benzydamine by molecular docking, pull-down assay and in vitro kinase assay. Specifically, benzydamine inhibited the growth of ESCC cells by inhibiting CDK2 activity and affecting downstream phosphorylation of MCM2, c-Myc and Rb, resulting in cell cycle arrest. Our study illustrates that benzydamine inhibits the growth of ESCC cells by downregulating the CDK2 pathway.
Subject(s)
Humans , Benzydamine , Esophageal Neoplasms/drug therapy , Esophageal Squamous Cell Carcinoma/drug therapy , Molecular Docking Simulation , Phosphorylation , Cell Proliferation , Cell Line, Tumor , Apoptosis , Cyclin-Dependent Kinase 2ABSTRACT
Objective:To investigate the diagnostic values of detections of human papillomavirus (HPV) DNA combined with peripheral blood cyclin A mRNA and cyclin-dependent kinase 2 (CDK2) mRNA for cervical squamous cell carcinoma.Methods:Eighty patients with cervical squamous cell carcinoma treated in Jiangyin Hospital of Traditional Chinese Medicine from January 2018 to October 2021 were selected as the research objects. Eighty patients with benign cervical lesions such as cervicitis treated in the same period were selected as the control. The levels of HPV-DNA in paraffin-embedded tissues of cervical squamous cell carcinoma were detected by gene chip, and the mRNA expression levels of cyclin A and CDK2 in peripheral blood monocytes were detected by reverse transcription polymerase chain reaction. The related factors of cervical squamous cell carcinoma were analyzed by multivariate logistic regression. Taking the results of pathological biopsy as the gold standard, the diagnostic efficacy of HPV-DNA, peripheral blood cyclin A mRNA and CDK2 mRNA single and combined detection for cervical squamous cell carcinoma were determined by receiver operating characteristic (ROC) curve.Results:The positive rate of HPV-DNA in patients with cervical squamous cell carcinoma was higher than that in the control group [75.00% (60/80) vs. 13.75% (11/80), P < 0.05]; the relative expressions of cyclin A mRNA and CDK2 mRNA in patients with cervical squamous cell carcinoma were 0.26±0.08 and 1.49±0.07, respectively, which were higher than those in the control group (0.11±0.03 and 1.14±0.06), and the differences were statistically significant (both P < 0.05). Multivariate logistic regression analysis showed that the number of induced abortions > 1 ( OR = 3.093, 95% CI 1.386-6.899, P = 0.021), the age of first birth ≤18 years old ( OR = 3.684, 95% CI 1.651-8.219, P = 0.013), the positive HPV-DNA ( OR = 4.125, 95% CI 1.849-9.202, P = 0.001), the increased relative expression of cyclin A mRNA in peripheral blood ( OR = 3.800, 95% CI 1.703-8.478, P = 0.006) and the increased relative expression of CDK2 mRNA in peripheral blood ( OR = 4.821, 95% CI 2.161-10.756, P = 0.008) were risk factors for the occurrence of cervical squamous cell carcinoma. ROC curve analysis showed that the area under the curve (AUC) of HPV-DNA, peripheral blood cyclin A mRNA and CDK2 mRNA in single and combined diagnosis of cervical squamous cell carcinoma were 0.769 (95% CI 0.700-0.838), 0.756 (95% CI 0.688-0.823), 0.755 (95% CI 0.689-0.820) and 0.827 (95% CI 0.766-0.888), respectively. Conclusions:HPV-DNA and the levels of cyclin A mRNA and CDK2 mRNA in peripheral blood can be used to assist in the diagnosis of cervical squamous cell carcinoma, and the combination of the three has high diagnostic efficiency.
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Objective@#To investigate the effect of transforming growth factor(TGF)-β1 stimulation on the proliferation of lung fibroblasts and the pathogenesis of bronchopulmonary dysplasia(BPD).@*Methods@#The lung fibroblasts of newborn rats were isolated, purified and identified on the 1st day after birth.The primary lung fibroblasts were stimulated by TGF-β1, and the protein expression of cyclin-dependent kinase-2(CDK2) and p27 were detected by immunohistochemistry and Western blotting.Real-time fluorescence quantitative polymerase chain reaction(PCR) was used to detect the mRNA expression of CDK2 and p27.CCK8 kit was used to detect cell proliferation, and cell cycle was detected by flow cytometry.@*Results@#The expression of CDK2 protein and mRNA in primary fibroblasts was increased after TGF-β1 stimulation in lung fibroblasts of newborn rats, accompanied by the decrease of p27 protein and mRNA.CCK8 showed that the value of A increased significantly, proliferation significantly increased the proportion of S phase cells at the same time, G0/G1 phase decreased.The percentage of S phase cells after stimulation with 0 μg/L, 10 μg/L TGF-β1 was 4.63%, 67.09%, respectively.The percentage of G0/G1 phase was 83.67% and 67.09%, respectively.The percentage of S phase cells was 7.64% and 9.11% respectively after stimulated with 10 μg/L TGF-β1 for 12 h and 48 h, respectively.The percentage of G0/G1 phase was 73.99% and 59.81% respectively.With the increase of TGF-β1 levels (0 μg/L, 5 μg/L and 10 μg/L), CDK2 protein and mRNA expression further increased; and the same level of TGF-β1 stimulation, stimulated for 12, 24 and 48 h, the expression of CDK2 protein and mRNA increased further, and increased significantly at 48 h (P<0.05 and <0.01 respectively). The levels of p27 protein and mRNA decreased further(all P<0.01).@*Conclusions@#TGF-β1 can up-regulate the expression of CDK2/p27 in primary lung fibroblasts of neonatal rats, accelerating cell cycle progression and over-proliferation of fibroblasts.
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Objective To evaluate the antitumor effect of dacarbazine (DTIC) on B16-F1 melanoma after CDK2 gene silencing.Methods Cultured B16-F1 melanoma cells were divided into 4 groups:control group receiving no treatment,CDK2-shRNA group infected with a recombinant lentivirus pUL-CDK2-shRNA,DTIC group cultured in 96-well plates followed 12 hours later by the treatment with 250 μmol/L DTIC,CDK2-shRNA + DTIC group infected with pUL-CDK2-shRNA followed 12 hours later by the treatment with 250 μmol/L DTIC.MTT assay was performed to evaluate the growth inhibition of B16-F1 melanoma cells,and coefficient of drug interaction (CDI) was calculated.AnnexinV-FITC/PI double staining was conducted to detect cell apoptosis.C57BL/6 mice were subcutaneously injected with B16-F1 cells at exponential growth phase into the right groin to establish melanoma-bearing mouse models.Twenty mouse models were randomly and equally divided into 4 groups:control mouse group injected with phosphate-buffered solution (PBS) into tumors,CDK2-shRNA mouse group injected with pUL-CDK2-shRNA into tumors,DTIC mouse group injected with DTIC into the abdominal cavity,and CDK2-shRNA + DTIC mouse group treated with pUL-CDK2-shRNA and DTIC.The animal experiment lasted 18 days,and the tumor growth curve was drawn.After 18-day treatment,all the mice were sacrificed,and tumors were isolated and weighed.The tumor growth inhibition rate was calculated,and the tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL).Results After 72-hour culture,compared with the control group,the CDK2-shRNA group,DTIC group,and CDK2-shRNA + DTIC group showed significantly decreased relative cell survival rates (40.6％ ± 2.8％,45.2％ ± 3.7％,28.7％ ± 2.1％,respectively;F =458.04,P ＜ 0.05),but significantly increased cell apoptosis rates (25.1％ ± 3.3％,15.6％ ± 2.2％,45.6％ ± 3.5％,respectively;F =115.46,P ＜ 0.05).Additionally,CDK2-shRNA + DTIC group showed significantly lower relative cell survival rates (P ＜ 0.01),but higher cell apoptosis rates (P ＜ 0.01) compared with the DTIC group.The CDI value was less than 0.7.On the sixth day after the in vivo treatment,the tumor volumes in the control mouse group,CDK2-shRNA mouse group,DTIC mouse group,and CDK2-shRNA + DTIC mouse group were (185.44 ± 68.97) mm3,(83.91 ± 14.33) mm3,(123.70 ± 20.85) mm3,and (34.54 ± 10.72) mm3 respectively.From then on,the CDK2-shRNA mouse group,DTIC mouse group,and CDK2-shRNA + DTIC mouse group showed significantly decreased tumor growth rates compared with the control mouse group (F =11.819,P ＜ 0.05),and the tumor growth rate was significantly lower in the CDK2-shRNA + DTIC mouse group than in the DTIC mouse group (P =0.04).The calculated tumor growth inhibition rates in the CDK2-shRNA mouse group,DTIC mouse group and CDK2-shRNA + DTIC mouse group were 52.2％,41.2％ and 86.4％ respectively.Compared with the control mouse group,the CDK2-shRNA mouse group,DTIC mouse group,and CDK2-shRNA + DTIC mouse group showed significantly increased tumor cell apoptosis indice (32.93％ ± 3.72％,21.62％ ± 3.54％,63.29％ ± 4.74％ respectively;F =222.25,P ＜ 0.05).Moreover,the tumor cell apoptosis index was significantly higher in the CDK2-shRNA + DTIC mouse group than in the DTIC mouse group (P ＜ 0.01).Conclusion CDK2 gene silencing can enhance the inhibitory effect of DTIC on the growth of melanoma,and show a synergistic effect with DTIC,likely by increasing the apoptosis of tumor cells.
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Objective To investigate the effect of cyclin-dependent kinase 2 (cdk2) recombinant lentivirus in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods Thirty-six adult SD rats were divided into control group, LPS model group and gene intervention group according to the random number table, with 12 rats per group.Rats with LPS-induced ALI were established by intratracheal injection of LPS.Saline solution (60 μL/kg) was injected in control group at the time point of 0, 24, 48 h respectively.Control-lentivirus (60 μL/kg) and cdk2 recombinant lentivirus (60 μL/kg) were injected respectively in LPS model group and gene intervention group at the time point of 0 h and 24 h.After 48 h, LPS (60 μL/kg) with isotonic saline solution were injected in both LPS model group and gene intervention group.Lung tissue samples from right-lower areas were collected at 24 h postinjury to evaluate the pathological changes with HE staining.Expressions of cdk2, clara cell secretory protein (CCSP), phospholipase A2(PLA2) and p-C/EBP β protein were detected by Western blot.Inflammatory factors of tumor necrosis factor-α(TNF-α), interleukin (IL)-1β, IL-6 and IL-10 in serum were measured with ELISA method.Results Inflammatory infiltration and damage to the alveolar structure were serious in LPS model group than control group, while inflammatory infiltration decreased significantly and alveolar structure tended to be normal in gene-intervention group.Expression of Cdk2 in control group (1.00±0.21) and LPS model group (0.93±0.17) were similar, but both were lower than that in gene intervention group (4.29±0.73) (P<0.05).Expression of CCSP in gene intervention group (3.19±0.38) was significantly higher than that in control group (1.00±0.20) and LPS model group (0.32±0.19) (P<0.05).Expression of PLA2 in LPS model group (4.49±0.51) was higher than that in control group (1.00±0.13) and gene intervention group (1.76±0.26) (P<0.05).Meanwhile, the variation of p-C/EBPβ concentration among the groups was similar to CCSP.Expression of TNF-α in LPS model group[(196.34±30.17)pg/ml] was higher than that in control group [(71.24±5.13)pg/ml] and gene intervention group[(86.32±11.02)pg/ml](P<0.05).Changes in IL-1β, IL-6 and IL-10 among the groups were similar to TNF-α.Conclusions Over-expression of Cdk2 plays a protective role for LPS-induced ALIby up-regulating CCSP and down-regulating inflammatory factors such as PLA2, TNF-α, IL-1β, IL-6 and IL-10, as may relate to the phosphorylation of C/EBPβ.
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AIM:To investigate the effects and mechanisms of microRNA-25 (miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1.METHODS: The abundance of miRNA-25 in different tis-sues was measured by RT-PCR.After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2 (CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respective-ly.RESULTS:miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05).O-ver-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the en-trance of the cells into S phase (P<0.05).Inverse results were obtained after down-regulation of miRNA-25 (P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-ex-pression of miRNA-25, but decreased after down-regulation of miRNA-25 (P<0.05).CONCLUSION: miRNA-25 en-hances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for trea-ting esophageal squamous-cell carcinoma.
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AIM:To investigate the effects of microRNA-193 (miR-193) on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells (MSCs).METHODS:Cultured rat MSCs were transfected with pre-miR-193 or anti-miR-193 to regulate the expression of miR-193.The proliferation of the MSCs after transfection was evaluated by MTS assay, colorimetric BrdU cell proliferation assay and Ki-67 immunostaining.Cell apoptosis was analyzed by flow cytometry with Annexin V/PI staining.The effect of miR-193 on the expression of cell cycle-related proteins was evaluated by qRT-PCR.RESULTS:Transfection of pre-miR-193 or anti-miR-193 regulated the expression of miR-193 in MSCs effectively . Over-expression of miR-193 significantly promoted the proliferation of MSCs ( P0.05).The result of qRT-PCR indicated miR-193 promoted the expression of cyclin-dependent kinase 2 ( CDK2 ) significantly ( P<0.01).CONCLUSION:miR-193 promotes the proliferation of MSCs possibly through the CDK 2 pathway.
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AIM:To investigate the efficiency of 2-methoxyestradiol (2-ME) as radiosensitizing agent for the treatment of lung cancer cells. METHODS:Cell line A549 and GLC-82 originated from human non-small cell lung cancer were cultured in vitro. Study group (2-ME in different concentrations) and control group without 2-ME were set up. Cell proliferation was measured by MTT assay that lung cancer cells were treated with 2-ME for 24 h,then the cells were exposed from 0 to 8Gy radiation,and the survival fraction was determined by clone forming test. Flow cytometry was used to measure the effects of 2-ME on cell cycle distribution. RESULTS:MTT assay showed minimum effective concentration value was 0.15625×10~(-6) mol/L in GLC-82 and 1.25×10~(-6) mol/L in A549 cells. Compared to control group,exposed GLC-82 cells or A549 cells to minimum effective concentration of 2-ME for 24 h before irradiation resulted in an enhancement of radiation. The protection enhancement factor was 1.98 and 2.06 in GLC-82 and A549 cells,respectively. Flow cytometry analysis of cell cycle progression demonstrated G_2/M phase arrest in both cells in a dose dependent manner. No obvious change of CDK2 activity in both GLC-82 cells and A549 cells was observed. CONCLUSION:2-ME enhances radiosensitivity by G_2/M phase arrest in the cell cycle.
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Aim: To evaluate the effect of Cdk7 silencing on the cell cycle control, the phosphorylation level changes of Cdk2 and pRb in human hepatoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel target for anticancer therapeutics. Method: Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA contents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flow cytometry and ultrastructural changes of cells were observed with transmission electron microscopy. Result The phosphorylation levels of pRb and Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner when the concentration was above 100 nmol·L-1. Indice of cells arrested in G0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P < 0.01); Characteristic apoptosis in Cdk7 ASODN treated groups were obvious under the transmission electron microscopy. Conclusion: Bioactivities and phosphorylation levels of pRb and Cdk2 decreased after Cdk7 silencing and thus induced obvious G0/G1 phases arrest and apoptosis in HepG2 cell culture in vitro, it is feasible to consider Cdk7 as a novel target for anticancer therapeutics.
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Aim To evaluate the effect of Cdk7 silencing on the c ell cycle control, the phosphorylation level changes of Cdk2 and pRb in human he patoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel ta rget for anticancer therapeutics.Method Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA c ontents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flo w cytometry and ultrastructural changes of cells were observed with transmission electron microscopy.Result The phosphorylation levels of pRb a nd Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner wh en the concentration was above 100 nmol?L -1. Indice of cells arrested in G 0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P