ABSTRACT
The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.
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Objective: To investigate the cell death-inducing effect of saikosaponin-d (SSD) on human hepatoma Hep3B cells and its potential mechanism. Methods: Hepatoma Hep3B cells were divided into five groups: blank control group, DMSO (vehicle) group, and three SSD treatment groups treated with various doses of SSD (5, 10, and 15 μmol/L). MTT assay was used to evaluate cell viability. Flow cytometer was employed to quantitatively detect the percentage of dead cells after Annexin V-FITC/PI double staining. Apoptosis was detected morphologically after Hoechst 33258 staining. The activity of caspase-3 apoptotic protease was determined by spectrophotometry. Cell morphologic changes were observed with an inverted microscope. Western blotting and Real-time PCR were employed to evaluate the expression levels of C/EBP homology (CHOP) protein and mRNA, respectively. Results: MTT assay showed that SSD inhibited the viability of human hepatoma Hep3B cells in concentration- and time-dependent manners. Sinomenine hydrochloride induced the death of Hep3B cells in a concentration-dependent manner indicated by flow cytometry. After staining with Hoechst 33258, the nuclei of SSD-treated cells showed nucleosomal agglutination, nucleosomal shrinkage and fragmentation under the fluorescence microscope, which are the characteristics of apoptotic cells. SSD significantly activated the key apoptotic executor caspase-3. The occurrence of paraptosis, characterized by extensive cytoplasmic vacuoles, was observed in SSD-treated cells under an inverted microscope. The pretreatment of a pancaspase inhibitor Z-VAD-FMK completely inhibited caspase-3 activity triggered by SSD, but only partially suppressed cell death and could not reduce the cytoplasmic vacuolation in SSD-treated cells. The protein and mRNA expressions of CHOP, a stress-inducible molecule, were upregulated by SSD, which could not be inhibited by Z-VAD-FMK. Conclusion: SSD can simultaneously induce caspase-dependent apoptosis and caspase-independent paraptosis in human hepatoma Hep3B cells. The upregulated expression of CHOP may be the mechanism involved in SSD-induced paraptosis.
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· AIM:To study whether autophagy and paraptosis were activated in retinal ganglion cells (RGCs) after acute high intraocular pressure (lOP) in an experimental rat model and to explore the possible underlying mechanisms.· METHODS:A total of 50 male Sprague-Dawley (SD)rats were randomly divided into normal control group,and 3d,1,4,8wk group after acute elevated intraocular pressure(IOP) (n =10 per group).Acute intraocular hypertension model was established by anterior chamber perfusion of normal saline in the right eye.The expression levels of microtubule-associated protein 1 light chain 3 (LC3) was measured by immumofluorescence method.To determine whether autophagy and paraptosis were activated.Retinal sections were examined by transmission electron microscopy (TEM).Autophagosomes and cytoplasmic vacuoles in the cytoplasm of RGCs were measured.· RESULTS:TEM analysis revealed that double-and multiple-membrane vacuoles containing electron-dense materials of autophagosomes were found in RGCs.The number of autophagosomes per 50μ m2 were 0.79 ± 0.43,2.14±0.36,2.29±0.47,1.57±0.51 and 1.21±0.43 in the normal control group and in acute IOP group at 3d,1wk,4wk,8wk,respectively.The number of autophagosomes markedly increased in the cytoplasm of RGCs at 3d,1wk,4wk,8wk groups than those in the normal control group (all at P＜ 0.05).LC3 positive expression was rarely detected in ganglion cell layer (GCL) in the normal control group and percentage of LC3 positive cells was 15.90％.Immumofluorescence analysis showed that the percentage of LC3 positive cells statistically increased in acute lOP groups when compared with control group (P＜0.05).The number of RGCs per 200μm in each group of acute lOP injury significantly decreased compared with the normal control group (P ＜ 0.05).Cytoplasmatic vacuolization were observed in RGCs at 3d after acute lOP injury and lasting to 8wk.TEM also revealed that a large number of cytoplasmic vacuoles were derived predominantly from the progressive swelling of mitochondria and/or endoplasmic reticulum (ER).· CONCLUSION:Autophagy and paraptosis participate in the death of RGCs under transiently elevated intraocular pressure.Different types of programmed cell death (PCD),coexistence of multiple cell death forms or a single cell death form,participates in the pathogenesis of acute elevation of intraocular pressure.
ABSTRACT
Objective To investigate whether paraptosis and autophagy have an effect on acute retinal ischemia-reperfusion injury (RIRI) in an experimental rat model that recapitulates features of acute hypertensive glaucoma and to explore the possible underlying mechanisms.Methods A total of 30 adult male Sprague-Dawley rats were randomly divided into RIRI group and control group.The acute RIRI model was induced with normal saline in the right eye of rats from the RIRI group by anterior chamber perfusion,while the rats in the control group left untreated.On day 1,day 3,day 7,day 28 after RIRI model establishment,the changes in morphology of retinal ganglion cells (RGCs) were observed by transmission electron microscopy (TEM),and the expression of microtubule-associated protein 1 light chain 3 (LC3) was measured by immumofluorescence methods.Results When compared with the control group,the number of cytoplasmic vacuoles predominantly derived from the progressive swelling of mitochondria and/or endoplasmic reticulum (ER) in RGCs were increased in the RIRI group from day 1 to day 28 by TEM.And ultra-structural analyses showed the double-or multiple-membrane autophagosomes were markedly accumulated in the cytoplasm of RGCs following acute RIRI.The average number of autophagic vacuoles in the cytoplasm of RGCs was 0.79 per 50 μm2 in the control group,and the average number of autophagosomes reached to a maximum on day 7 after acute RIRI at 2.29 per 50 μm2,which was statistically significant compared with the control group (P ＜ 0.05).Compared to the control group,LC3 expression in the cytoplasm of RGCs was up-regulated on day 1 after acute RIRI,which sustained throughout the experimental period.The percentage of LC3 positive cells in the retinal ganglion cell layer was 15.90％ in the control group,and the data was 46.95％ and 52.30％ on day 1 and day 28 after RIRI,respectively,both which were statistically significant compared with the normal control group (both P ＜ 0.05).Conclusion Both paraptosis and autophagy participate in death of RGCs after acute RIRI.Programmed cell death in different cells,either coexistence of multiple-cell death form or a single-cell death form,participates in the pathogenesis of acute RIRI.
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Objective To detect the expression profile of transcription factor C/EBPβ in human immortalized normal hepatic cell lines and hepatocellular carcinoma cell lines so as to determine the correlation between C/EBP3 with cell death mediated by endoplasmic reticulum stress in hepatocellular cells.Methods We cultured the human immortalized normal hepatic cells lines HHL5 and HL7702 and hepatocellular carcinoma cell lines SMMC7721;Bel7402,HepG2 and Hep3B.Hep3B cells were used as the cell model in tunicamycin-induced endoplasmic reticulum stress.Cellular morphology was observed under an inverted optical microscope.MTT assay was used to assess the inhibition of cell growth.To detect cell apoptosis,the cells were dyed with Hoechst 33258 and observed using a fluorescence microscope.RToPCR and Western blotting were used to detect the expression of at mRNA and protein levels,respectively.Results We found that normally the mRNA and protein isoform of C/EBPβ,C/EBPβ-1,were both expressed in all of the four hepatocellular cell lines and the two immortalized normal hepatic cell lines,while C/EBPβ protein isoform C/EBPβ-3 was only expressed in the two immortalized normal hepatic cell lines.Tunicamycin increased the expressions of both mRNA and protein of C/EBPβ in Hep3B cells and the increase of protein isoform C/EBPβ-3 was the most remarkable.In Hep3B cells,cell death was induced by tunicamycin through endoplasmic reticulum stress activity.Apoptosis as well as paraptosis was observed in tunicamycin-induced cell death.Conclusion C/EBPβ-3,one of the protein isoforms of C/EBPβ,is only expressed in normal hepatic cell lines,but not in hepatocellular cell lines.C/EBPβ is involved in cell death mediated by endoplasmic reticulum stress activity in hepatocellular carcinoma cells.