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Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer’s disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
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Objective Little is known about the effect of RNAi on mitochondrial apoptotic pathways. This study aims to explore the effects of the Survivin shRNA-APC double-gene on colon cancer mitochondrial apoptosis pathway-related factors survivin,cytochrome C (Cytc),second mitochondria-derived activator of caspases (Smac),and cysteine aspartic acid specific protease 9 (Caspase-9) as well as on the apoptosis of colon cancer transplanted tumor (CCTT) cells. Methods Thirty nude mice were randomly divided into five groups of equal number,Survivin shRNA-APC double-gene,survivin shRNA,APC,empty vector and blank transfection. The CCTT model was established in the nude mice by subcutaneous injection of the colon cancer cell strains stably transfected with the Survivin shRNA-APC double-gene,survivin shRNA,APC,an empty vector and HT-29,respectively,into the mid-posterior part of the left armpit of the nude mice. The rate of tumor growth inhibition was calculated by measuring the volume and weight of the CCTTs in the nude mice. The mRNA and protein expressions of survivin,Cytc,Smac and Caspase-9 in the tumor tissue were detected by real time PCR and immunohistochemistry,respectively,and the apoptosis rate of the CCTT cells was detected by TUNEL. Results The model of CCTT was successfully established in the nude mice. Com-pared with the empty vector and blank transfection groups,the mice in the double-gene,survivin shRNA and APC groups showed sig-nificantly decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). In comparison with the survivin shRNA and APC groups,the double-gene group exhibited remarkably decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). The mRNA and pro-tein expressions of survivin were significantly lower while those of Cytc,Smac and Caspase-9 markedly higher in the double-gene,sur-vivin shRNA and APC groups than in the empty vector and blank transfection groups (P<0.05),the former even lower (P<0.05) and the latter even higher in the double-gene than in the survivin shRNA and APC groups (P<0.05). The apoptosis rate of the CCTT cells was significantly increased in the double-gene ([56.78±3.04]%),survivin shRNA ([33.61±2.02]%) and APC groups ([30.16± 1.72]%) as compared with the empty vector ([10.05±0.42]%) and blank transfection groups ([9.87±0.30])% (P<0.05),even higher in the double-gene group than in the survivin shRNA and APC groups (P<0.05). Conclusion The Survivin shRNA-APC double-gene may induce apoptosis of colon cancer transplanted tumor cells by down-regulating the expression of the apoptosis inhibitor survivin,upregulating the expressions of Cytc,Smac and Caspase-9,and suppressing the growth of the colon transplanted tumor,with more significant abilities than a single gene in regulating apoptosis-related factors,inducing cell apoptosis and inhibiting the growth of the transplanted tumor.
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AIM:To investigate the effects of survivin inhibitor YM155 {4,9-dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide} on the apoptosis, mito-chondrial membrane potential (Δψm) and cytochrome C (Cyt C) of retinoblastoma Y79 cells, and to analyze the mitochon-drial mechanisms of apoptosis .METHODS:Y79 cells were cultured in vitro and treated with YM155 at concentrations of 0, 0.5, 1, 2, 4 and 8 nmol/L.The cells in control group were treated without YM 155.The proliferation of Y79 cells were measured by CCK-8 assay and bromodeoxyuridine ( BrdU) labeling assay .Y79 cells were randomly divided into 4 groups:control group ( with equal volume of RPMI-1640 nutrient medium ) , positive control group ( 10 nmol/L topotecan ) , low-dose (1 nmol/L) YM155 group and high-dose (2 nmol/L) YM155 group.The effects of YM155 on the apoptosis, the changes of Δψm , the mitochondrial distribution and the protein level of Cyt C in the Y 79 cells were evaluated by flow cytom-etry with Annexin V-FITC/PI staining, JC-1 staining, immunofluorescence analysis and Western blot , respectively.RE-SULTS:Compared with control group , YM155 significantly inhibited the proliferation of Y 79 cells and induced apoptosis (P<0.05).YM155 significantly reduced Δψm of the Y79 cells, promoted Cyt C which released from mitochondria to the cytosol and reduced the protein level of Cyt C in the mitochondria (P<0.05).CONCLUSION:YM155 inhibits Y79 cell proliferation and induces apoptosis , and the possible mechanisms may be involved in the mitochondrium-mediated apoptotic pathway .
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<p><b>OBJECTIVE</b>To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.</p><p><b>METHODS</b>Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.</p><p><b>RESULTS</b>Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).</p><p><b>CONCLUSION</b>Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.</p>
Subject(s)
Animals , Rats , Apoptosis , Cadmium , Toxicity , Caspases , Metabolism , Mitochondria , Neurons , PC12 Cells , Proto-Oncogene Proteins c-akt , Metabolism , Rats, Sprague-Dawley , Reactive Oxygen Species , Metabolism , Signal Transduction , TOR Serine-Threonine Kinases , MetabolismABSTRACT
Objective To investigate the expression of AIF, CYT C, PAF-1, caspase-3, and XIAP in Sprague-Dawley rats with spontaneous mammary neoplasms.Methods One-hundred and thirty 3-4-week old SPF Spargue-Dawley rats (♀∶♂=1∶1) were fed in a specific pathogen free (SPF) breeding barrier for 60 weeks.The occurrence of spontane-ous breast tumors was recorded and histopathology was performed to identify the types of tumors.The rats were divided into 3 groups:rats with normal breast tissue ( group I) , with benign tumors ( group II) and with malignant tumors ( group III) . The expression of AIF, CYT C, APAF-1, caspase-3 and XIAP proteins and mRNAs were detected by immunhistochemistry ( IHC) and RT-PCR assay.Results Among these 130 SD rats, 14 rats were observed having spontaneous mammary neo-plasms with the incidence rate of 10.77%(14/130).In these neoplasm cases, 7 cases were mammary fibroadenomas, 7 cases of breast carcinoma, both with an incidence rate of 5.38%.Immunohistochemistry showed that, compared with the group I, the positive expressions of AIF, APAF-1, caspase-3 were decreased significantly (P<0.01), and the CYT C and XIAP expressions were significantly increased in the group II.The positive expression of all genes except XIAP was de-creased in the group III(P<0.01).Compared with the group II, APAF-1 and XIAP were significantly higher in the group III (P<0.01), and the positive expression of AIF, Cyt C, and caspases-3 were significantly decreased (P<0.01).In the results of RT-PCR assay, except APAF-1 which showed significant correlation with the results of immunohistochemistry ( P<0.05 ) , all the others showed an extremely significant correlation with immunohistochemical results ( P <0.01 ) . Conclusions Mammary tumors are most common spontaneous neoplasms in SD rats.Abnormal expression of mitochondrial apoptotic pathway-related factors AIF, CytC, APAF-1, caspase-3, and XIAP are correlated with the carcinogenesis and de-velopment of breast tumors.
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Background:Clinical and epidemiological studies revealed that estrogen replacement therapy was associated with a significant reduction in risk of colorectal cancer in postmenopausal women.In our previous studies,estrogen increased the expression of mismatch repair (MMR)gene MLH1 in colonic cancer cells,and re-expression of MLH1 in MLH-deficient colonic cancer cells significantly increased the estrogen-induced apoptosis.Aims:To investigate the signaling pathway implicated in the MLH1-mediated apoptosis in colonic cancer cells induced by estrogen and the roles of p53 and its related genes in this apoptotic pathway.Methods:Plasmid containing wild type human MLH1 (hMLH1)full length cDNA was transfected into MLH1-deficient human colonic cancer cell line HCT116.By using HCT116 cells transfected with empty plasmid as controls,the apoptotic DNA ladder was determined by electrophoresis and the expressions of p53 and other apoptosis-related proteins were assessed by Western blotting under the condition with or without estrogen stimulation. Results:17β-estradiol (E2 )at the concentration of 10 -8 mol/L induced significant apoptosis in HCT116 cells transfected with hMLH1.In HCT116 cells transfected with hMLH1 and stimulated with E2 (group D),the protein expressions of caspase-3,caspase-9,p53,Bax and cytoplasmic cytochrome C increased significantly when compared with HCT116 cells stimulated with E2 only (group B);expressions of the abovementioned proteins were also higher in group D than in group C (transfected with hMLH1 only).Conclusions:MMR gene MLH1 is involved in estrogen-induced apoptosis of human colonic cancer cell line HCT116 by activating p53 signaling and mitochondrial apoptotic pathway.
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AIM: To observe the effect of puerarin on myocardial injury according to the time of occurrence of myocardial injury in the development of type 2 diabetic mice.METHODS: The serum levels of glucose(GLU),triglyceride(TG),total cholesterin(TC),low density lipoprotein-cholesterol(LDL-C) and high density lipoprotein-cholesterol(HDL-C) in 17-week,20-week,24-week,28-week KKAy mice were detected by automatic biochemical methods.The apoptotic percentage of cardiomyocytes was examined by flow cytometry.The expressions of bax and bcl-2 mRNA in cardiomyocytes were detected by RT-PCR.Caspase-3 expression in cardiomyocytes was determined by immunohistochemical staining.RESULTS: Compared to normal control mice,not only GLU level increased,but also the levels of TG,TC,LDL-C,HDL-C in 20-week,24-week and 28-week KKAy mice increased apparently(P