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Objective:To observe the effects of electroacupuncture preconditioning on the autophagy-related pathway protein kinase B (Akt)/mammalian target of rapamycin (mTOR) in myocardial tissue of rats with myocardial ischemia reperfusion injury (MIRI); To investigate the protective mechanism of "Neiguan"(PC 6) on myocardial injury.Methods:Totally 48 SD rats were randomly divided into blank group, sham-operation group, model group and Neiguan group ( n=12 in each group). The Neiguan group was applied to bilateral "Neiguan"(PC 6) by electroacupuncture for 30 min, once daily for consecutive 7 days before model replication. Except in the blank group, the MIRI model was established by ligation of the descending anterior branch of the left coronary artery in the rest groups after the intervention. The histomorphological changes in the myocardium of the rats were observed by HE staining, and the expression levels of Akt, phosphorylated Akt (p-Akt), mTOR and phosphorylated mTOR (p-mTOR) in the myocardium were measured by protein immunoblotting. The ratio of p-Akt/Akt and p-mTOR/mTOR was calculated. Results:In the blank group, the myocardial fibres were arranged regularly and neatly, and no inflammatory cell infiltration or haemorrhage was seen in the interstitium; in the sham-operation group, the arrangement of myocardial fibers was slightly irregular, no rupture was found, and a small amount of myocardial fiber gap was slightly enlarged; in the model group, the distribution of myocardial fibers was disordered, hypertrophic cardiomyocytes increased, some mitochondria were red and swollen or the outer membrane was ruptured, and inflammatory infiltration and hemorrhage were seen in the interstitium; the extent of myocardial lesions in the Neiguan group was less than that in the model group, with a small amount of interstitial hemorrhage and inflammatory cell infiltration. There was no statistical significance in the levels of Akt and mTOR in the myocardial tissues of the rats in each group ( P>0.05); compared with the sham-operation group, the levels of p-Akt, p-mTOR and p-Akt/Akt, p-mTOR/mTOR in the model group decreased ( P<0.01); compared with the model group, the levels of p-Akt, p-mTOR and p-Akt/Akt, p-mTOR/mTOR in the Neiguan group increased ( P<0.01). Conclusion:Electroacupuncture preconditioning may inhibit excessive autophagy by activating the Akt/mTOR pathway in cardiomyocytes of MIRI rats, thereby exerting a protective effect on the myocardium.
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Objective:To observe the effect of Tangtong formula on the expression of autophagy pathway proteins PI3K, Akt and mTOR in sciatic nerve of DPN rats, and to explore its mechanism. Methods:There were 60 male SD rats, 15 of which were randomly selected as the normal group, and the other rats were used to establish DPN model with STZ + ischemia-reperfusion method. Then they were divided into model group, Tangtong formula low-dose group and Tangtong formula high-dose group, with 15 rats in each group with random number table method. 36.67 g/kg Tangtong formula was administered by gavage in the high-dose group and 18.33 g/kg Tangtong formula was administered by gavage in the low-dose group, once a day. After 8 weeks of continuous gavage, the conduction velocity of sciatic nerve was detected. The mRNA and protein expression levels of PI3K, Akt and mTOR were detected by PCR and Western blot. The structure of sciatic nerve fibers was observed by HE staining. Results:Compared with the model group, the motor nerve conduction velocity, sensory nerve conduction velocity, muscle compound action potential and sensory nerve action potential in the low-dose Tangtong formula group and high-dose Tangtong formula group were increased ( P<0.05). The expression of PI3K mRNA(6.05±0.18, 3.36±0.29 vs. 11.57±1.93), Akt mRNA(1.26±0.13, 0.64±0.04 vs. 1.86±0.06), mTOR mRNA(1.82±0.11, 0.92±0.06 vs. 2.68±0.18) of sciatic nerve in rats of the low-dose and high-dose group were increased ( P<0.05). The expression of PI3K(0.40±0.00, 0.19±0.02 vs. 0.61±0.03), Akt(0.64±0.02, 0.45±0.01 vs. 0.83±0.02), mTOR(0.17±0.01, 0.09±0.00 vs. 0.34±0.01)of sciatic nerve in rats of the low-dose and high-dose group were increased ( P<0.05). The model group's nerve fibers were loose and swollen, myelin sheath became thin, and the axis Atresia, the neuromorphology of the low-dose and high-dose group tended to be normal, and the morphology of myelin sheath and axon were better. Conclusions:Tangtong formula could improve the conduction velocity and potential amplitude of sciatic nerve in DPN rats, reduce nerve injury and demyelinating changes, improve axon morphology and protect nerve fiber structure. Its mechanism might be related to activating PI3K/Akt/mTOR signal pathway and inhibiting excessive autophagy.
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Diabetic mellitus (DM) is a common degenerative chronic metabolic disease often accompanied by severe cardiovascular complications (DCCs) as major causes of death in diabetic patients with diabetic cardiomyopathy (DCM) as the most common DCC. The metabolic disturbance in DCM generates the conditions/substrates and inducers/triggers and activates the signaling molecules and death executioners leading to cardiomyocyte death which accelerates the development of DCM and the degeneration of DCM to heart failure. Various forms of programmed active cell death including apoptosis, pyroptosis, autophagic cell death, autosis, necroptosis, ferroptosis and entosis have been identified and characterized in many types of cardiac disease. Evidence has also been obtained for the presence of multiple forms of cell death in DCM. Most importantly, published animal experiments have demonstrated that suppression of cardiomyocyte death of any forms yields tremendous protective effects on DCM. Herein, we provide the most updated data on the subject of cell death in DCM, critical analysis of published results focusing on the pathophysiological roles of cell death, and pertinent perspectives of future studies.
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Abstract Background: Viruses are being used as alternative and complementary tools for treating cancers. Oncolytic viruses exhibit tumor tropism, ability to enhance anti-tumor immunity and ability to be used in combination with conventional chemotherapy and radiotherapy. We have recently selected some rotavirus isolates which are adapted to efficiently infect and kill tumor cell lines. Aim: We tested five tumor cell-adapted rotavirus isolates for their ability to infect the human adenocarcinoma cell line MCF-7. Methods: Cell surface membrane-associated proteins mediating virus particle attachment were characterized using ELISA, immunoprecipitation, FACS analysis, and antibody blocking. Results: It was found that heat shock proteins (HSPs) such as Hsp90, Hsp70, Hsp60, and Hsp40 are expressed on the cell surface forming complexes with protein disulfide isomerase (PDI), integrin β3, and heat shock cognate protein 70 (Hsc70) in lipid raft microdomains. Interaction of rotavirus isolates with these cellular proteins was further confirmed by a competition assay and an inhibition assay involving the HSPs tested. Conclusion: Our findings suggest that the tumor cell-adapted rotavirus isolates studied here offer a promising tool for killing tumor cells, thus encouraging further research into this topic, including animal models.
Resumen Antecedentes: Los virus se utilizan como herramientas alternativas y complementarias para el tratamiento del cáncer. Los virus oncolíticos exhiben tropismo por tumores, capacidad para intensificar la inmunidad antitumoral y la capacidad para utilizarse en combinación con quimioterapia y radioterapia convencionales. Recientemente, hemos seleccionado algunos aislamientos de rotavirus que están adaptados para infectar y eliminar de manera eficiente líneas de células tumorales. Objetivo: Se ensayaron cinco aislamientos de rotavirus adaptados a células tumorales para determinar su capacidad para infectar la línea celular de adenocarcinoma humano MCF-7. Métodos: Las proteínas asociadas a la membrana de la superficie celular que median la unión de partículas de virus se caracterizaron mediante ELISA, inmunoprecipitación, análisis FACS y bloqueo de anticuerpos. Resultados: Se encontró que las proteínas de choque térmico (HSPs) como Hsp90, Hsp70, Hsp60 y Hsp40 se expresan en la superficie celular formando complejos con la proteína disulfuro isomerasa (PDI), la integrina β3 y la proteína análoga de choque térmico 70 (Hsc70) en microdominios lipídicos (rafts). La interacción de los aislamientos de rotavirus con estas proteínas celulares se confirmó adicionalmente mediante un ensayo de competición y un ensayo de inhibición que incluía las HSP ensayadas. Conclusión: Nuestros hallazgos sugieren que los aislamientos de rotavirus adaptados a las células tumorales estudiados aquí ofrecen una herramienta prometedora para eliminar las células tumorales, lo que estimula más investigaciones sobre este tema, incluidos los modelos animales.
Subject(s)
Humans , Adenocarcinoma , Rotavirus , Oncolytic Viruses , Heat-Shock Proteins , Adenocarcinoma/therapy , HSC70 Heat-Shock Proteins , MCF-7 CellsABSTRACT
Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which could result in autophagic cell death (ACD). Though novel combination chemotherapy of autophagy inducers with chemotherapeutic agents is extensively investigated, nanomedicine-based combination therapy for ACD remains in infancy. In attempt to actively trigger ACD for synergistic chemotherapy, here we incorporated autophagy inducer rapamycin (RAP) into 7pep-modified PEG-DSPE polymer micelles (7pep-M-RAP) to specifically target and efficiently priming ACD of MCF-7 human breast cancer cells with high expression of transferrin receptor (TfR). Cytotoxic paclitaxel (PTX)-loaded micelle (7pep-M-PTX) was regarded as chemotherapeutic drug model. We discovered that with superior intracellular uptake and more tumor accumulation of micelles , 7pep-M-RAP exhibited excellent autophagy induction and synergistic antitumor efficacy with 7pep-M-PTX. Mechanism study further revealed that 7pep-M-RAP and 7pep-M-PTX used in combination provided enhanced efficacy through induction of both apoptosis- and mitochondria-associated autophagic cell death. Together, our findings suggested that the targeted excess autophagy may provide a rational strategy to improve therapeutic outcome of breast cancer, and simultaneous induction of ACD and apoptosis may be a promising anticancer modality.
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@#Objective To observe the autophagy of 87-MG and U251 glioma cells induced by ceramide and explore the possible mechanism. Methods The viability and apoptosis of 87-MG and U251 cells were detected by MTT assay and flow cytometry, respectively. Autophagic- related protein expressions of LC3B /LC3A and Beclin-1 were determined by Western blotting. The activation of JNK/c-Jun signaling pathway induced by ceramide with or without the treatment of JNK specific inhibitor SP600125 was also measured. Results 24 hours after treatment of ceramide, the growth of 87-MG and U251 cells was significantly inhibited time-dependently (P<0.05); and the number of autophagic cells increased dose-dependently (P<0.05). The levels of LC3B/LC3A and Beclin-1 significantly increased after ceramide treatment (P<0.05). JNK signaling pathway was activated in the 87-MG and U251 cells and the phosphorylation of c-Jun also increased after ceramide treatment. This activation of autophagy could be reversed by the pre-treatment of SP600125. Conclusion Ceramide may induce autophagy in 87-MG and U251 glioma cells and the mechanism may be related to the activation of JNK/c-Jun signaling pathway.
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Objective To observe the autophagy of 87-MG and U251 glioma cells induced by ceramide and explore the possible mecha-nism. Methods The viability and apoptosis of 87-MG and U251 cells were detected by MTT assay and flow cytometry, respectively. Autoph-agic-related protein expressions of LC3B/LC3A and Beclin-1 were determined by Western blotting. The activation of JNK/c-Jun signaling pathway induced by ceramide with or without the treatment of JNK specific inhibitor SP600125 was also measured. Results 24 hours after treatment of ceramide, the growth of 87-MG and U251 cells was significantly inhibited time-dependently (P<0.05);and the number of au-tophagic cells increased dose-dependently (P<0.05). The levels of LC3B/LC3A and Beclin-1 significantly increased after ceramide treat-ment (P<0.05). JNK signaling pathway was activated in the 87-MG and U251 cells and the phosphorylation of c-Jun also increased after ce-ramide treatment. This activation of autophagy could be reversed by the pre-treatment of SP600125. Conclusion Ceramide may induce au-tophagy in 87-MG and U251 glioma cells and the mechanism may be related to the activation of JNK/c-Jun signaling pathway.
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Objective To provide more convincing evidences and experimental data for exploring vanillin derivative BVAN08,6-bromine-5-hydroxy-4-methoxy-benzaldehyde,as a new anticancer drug,and to investigate the effect on the growth,radiosensitization of human glioma cell line U-251 and the relative mechanism.Methods The effect of BVAN08 on cell proliferation of U-251 and radiosensitivity to 60Co γ-rays (irradiation dose rate 2.3 Gy/min) were analyzed with MTT and colony-forming ability assay.Change in cellular morphology was observed by using light microscope.Change in cell cycle and apoptosis was detected with flow cytometry.The autophagy was observed by using TEM (irradiation dose rate is transmission electron microscope).DNA-PKcs protein level was detected through Western blot analysis.Results BVAN08 exhibited a dose- and time-dependent inhibition on the proliferation of U-251 cells during the concentration range of 10-100 mol/L (t = 1.83-3.07,P ＜ 0.05).IC50 at 48 h and 72 h after administration with BVAN08 were 55.3 and 52.7 mol/L,respectively.Obvious G2/M arrest was induced in U-251 cells after 4 h administration with BVAN08,and reached peak at 12 h.The G2/M population reached 63.3% in U-251 cells after 12 h administration of 60 μmol/L BVAN08 and kept increasing with the time,while both apoptosis and autophagic cell death were induced.The most effective radiosensitization time for BVAN08 treatment was 12 h before irradiation.The enhancement ratio of radiosensitivity was 3.14 for 20 μmol/L of BVAN08 12 h before 2 Gy irradiation.Conclusions BVAN08 can nduce apoptosis as well as autophygic cell death of U-251 cells,and sensitize U-251 cells.The mechanism of its radiosensitizing effect might be associated with the induction of G2/M arrest and inhibition of DNA-PKcs expression.BVAN08 seemed to be a romising radiosensitizing anticancer drug.