BACKGROUND Necroptosis has emerged as a novel molecular pathway that can be targeted by chemotherapy agents in the treatment of cancer.OSW-1,which is derived from the bulbs of Ornithogalum saundersiae Baker,exerts a w...BACKGROUND Necroptosis has emerged as a novel molecular pathway that can be targeted by chemotherapy agents in the treatment of cancer.OSW-1,which is derived from the bulbs of Ornithogalum saundersiae Baker,exerts a wide range of pharmaco-logical effects.AIM To explore whether OSW-1 can induce necroptosis in colorectal cancer(CRC)cells,thereby expanding its range of clinical applications.METHODS We performed a sequence of functional experiments,including Cell Counting Kit-8 assays and flow cytometry analysis,to assess the inhibitory effect of OSW-1 on CRC cells.We utilized quantitative proteomics,employing tandem mass tag label-ing combined with liquid chromatography-tandem mass spectrometry,to analyze changes in protein expression.Subsequent bioinformatic analysis was conducted to elucidate the biological processes associated with the identified proteins.Transmission electron microscopy(TEM)and immunofluorescence studies were also performed to examine the effects of OSW-1 on necroptosis.Finally,western blotting,siRNA experiments,and immunoprecipitation were employed to evaluate protein interactions within CRC cells.RESULTS The results revealed that OSW-1 exerted a strong inhibitory effect on CRC cells,and this effect was accompanied by a necroptosis-like morphology that was observable via TEM.OSW-1 was shown to trigger necroptosis via activation of the RIPK1/RIPK3/MLKL pathway.Furthermore,the accumulation of p62/SQSTM1 was shown to mediate OSW-1-induced necroptosis through its interaction with RIPK1.CONCLUSION We propose that OSW-1 can induce necroptosis through the RIPK1/RIPK3/MLKL signaling pathway,and that this effect is mediated by the RIPK1-p62/SQSTM1 complex,in CRC cells.These results provide a theoretical foundation for the use of OSW-1 in the clinical treatment of CRC.展开更多
Objective: This study was designed to evaluate whether p62/SQSTM1 (hereafter referred to as p62) is involved in the immune response of macrophages against challenge byCandida albicans (C. albicans).Methods: We culture...Objective: This study was designed to evaluate whether p62/SQSTM1 (hereafter referred to as p62) is involved in the immune response of macrophages against challenge byCandida albicans (C. albicans).Methods: We cultured bone marrow-derived macrophages (BMDMs) to investigate the immune response to challenge byC. albicans. The p62 gene was knocked down by transfection with p62 small interfering RNA (siRNA) in the p62 siRNA group. BMDMs transfected with nonsense siRNA served as the negative control (NC) group. These two groups of BMDMs were challenged withC. albicans in vitro. We detected p62 expression through quantitative reverse transcription PCR and western blotting. The phagocytosis ability of BMDMs was evaluated by flow cytometry and microscopic examination using an Olympus FV1000 laser scanning confocal microscope. Moreover, we determined the level of reactive oxygen species (ROS) in BMDMs. The mRNA levels of proinflammatory cytokines were determined by quantitative reverse transcription PCR.Results: After stimulation byC. albicans, the relative expression of p62 mRNA was increased in a dose-dependent manner, the relative expression of p62 and the ratio of BMDMs toC. albicans is 1.893 ± 0.2156 (1:1,P < 0.05), 2.873 ± 0.4787 (1:3,P < 0.05) and 3.556 ± 0.2892 (1:5,P < 0.01). The p62 protein level was also increased. After transfection with p62 siRNA, the mRNA and protein levels of p62 were significantly decreased in BMDMs (P < 0.05). After 0.5, 1 and 2 hours of co-culture of BMDMs withC. albicans, flow cytometry showed that the phagocytosis rates ofC. albicans by BMDMs were significantly lower in the p62 siRNA group than in the NC group (39.70 ± 1.69%vs. 55.23 ± 0.72%, 46.70 ± 0.89%vs. 60.80 ± 1.78%, 51.90 ± 0.98%vs. 64.43 ± 2.0%, respectively, allP < 0.05). Consistent results were seen in the production of ROS (4269 ± 392.6vs. 13426 ± 1859.7, 4967 ± 721.2vs. 13687 ± 2611.2, 7647 ± 1950.0vs. 17719 ± 1814.2, respectively, allP < 0.05). The ROS levels were higher in BMDMs of the NC group than in BMDMs transfected with p62 siRNA at 0.5, 1, and 2 hours after treatment withC. albicans. BMDMs was co-cultured withC. albicans for 4 and 12 hours, the mRNA levels of interleukin-1β and interleukin-18 in NCs were also higher than p62 siRNA group, interleukin-1β: (6.14 ± 1.63vs. 12.12 ± 0.54, 8.81 ± 0.86vs. 26.2 ± 4.67, respectively, allP < 0.05), IL-18: (0.38 ± 0.02vs. 0.97 ± 0.06, 0.44 ± 0.02vs. 2.23 ± 0.46, respectively, allP < 0.05).Conclusion: p62 plays an important role in the process of phagocytosis in BMDMs challenged byC. albicans through ROS production and expression of proinflammatory cytokines.展开更多
Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditional...Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditionally mutations manifesting in the ubiquitin-associated domain of p62 were associated with Paget’s disease of bone,mutations affecting all functional domains of p62 have now been identified in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.p62 is a multifunctional protein that facilitates protein degradation through autophagy and the ubiquitin-proteasome system,and also regulates cell survival via the Nrf2 antioxidant response pathway,the nuclear factor-kappa B signaling pathway and apoptosis.Dysfunction in these signaling and protein degradation pathways have been observed in amyotrophic lateral sclerosis and frontotemporal lobar degeneration,and mutations that affect the role of p62 in these pathways may contribute to disease pathogenesis.In this review we discuss the role of p62 in these pathways,the effects of p62 mutations and the effect of mutations in the p62 modulator TANK-binding kinase 1,in relation to amyotrophic lateral sclerosis-frontotemporal lobar degeneration pathogenesis.展开更多
Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively d...Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.展开更多
基金Supported by the Natural Science Foundation of Liaoning Province,No.2022-MS-330and Key Projects in Liaoning Province,No.2020JH2/10300046.
文摘BACKGROUND Necroptosis has emerged as a novel molecular pathway that can be targeted by chemotherapy agents in the treatment of cancer.OSW-1,which is derived from the bulbs of Ornithogalum saundersiae Baker,exerts a wide range of pharmaco-logical effects.AIM To explore whether OSW-1 can induce necroptosis in colorectal cancer(CRC)cells,thereby expanding its range of clinical applications.METHODS We performed a sequence of functional experiments,including Cell Counting Kit-8 assays and flow cytometry analysis,to assess the inhibitory effect of OSW-1 on CRC cells.We utilized quantitative proteomics,employing tandem mass tag label-ing combined with liquid chromatography-tandem mass spectrometry,to analyze changes in protein expression.Subsequent bioinformatic analysis was conducted to elucidate the biological processes associated with the identified proteins.Transmission electron microscopy(TEM)and immunofluorescence studies were also performed to examine the effects of OSW-1 on necroptosis.Finally,western blotting,siRNA experiments,and immunoprecipitation were employed to evaluate protein interactions within CRC cells.RESULTS The results revealed that OSW-1 exerted a strong inhibitory effect on CRC cells,and this effect was accompanied by a necroptosis-like morphology that was observable via TEM.OSW-1 was shown to trigger necroptosis via activation of the RIPK1/RIPK3/MLKL pathway.Furthermore,the accumulation of p62/SQSTM1 was shown to mediate OSW-1-induced necroptosis through its interaction with RIPK1.CONCLUSION We propose that OSW-1 can induce necroptosis through the RIPK1/RIPK3/MLKL signaling pathway,and that this effect is mediated by the RIPK1-p62/SQSTM1 complex,in CRC cells.These results provide a theoretical foundation for the use of OSW-1 in the clinical treatment of CRC.
基金The study was supported by the National Natural Science Foundation of China(Nos.82103749,81773338,and 82173432)ChineseAcademy ofMedicalSciencesMedicine andHealthTechnology InnovationProject(No.2017-I2M-1-017)+1 种基金Natural Science Foundation of Jiangsu Province(No.BK20190144)the Nanjing Incubation Program for National Clinical Research Center(No.2019060001).
文摘Objective: This study was designed to evaluate whether p62/SQSTM1 (hereafter referred to as p62) is involved in the immune response of macrophages against challenge byCandida albicans (C. albicans).Methods: We cultured bone marrow-derived macrophages (BMDMs) to investigate the immune response to challenge byC. albicans. The p62 gene was knocked down by transfection with p62 small interfering RNA (siRNA) in the p62 siRNA group. BMDMs transfected with nonsense siRNA served as the negative control (NC) group. These two groups of BMDMs were challenged withC. albicans in vitro. We detected p62 expression through quantitative reverse transcription PCR and western blotting. The phagocytosis ability of BMDMs was evaluated by flow cytometry and microscopic examination using an Olympus FV1000 laser scanning confocal microscope. Moreover, we determined the level of reactive oxygen species (ROS) in BMDMs. The mRNA levels of proinflammatory cytokines were determined by quantitative reverse transcription PCR.Results: After stimulation byC. albicans, the relative expression of p62 mRNA was increased in a dose-dependent manner, the relative expression of p62 and the ratio of BMDMs toC. albicans is 1.893 ± 0.2156 (1:1,P < 0.05), 2.873 ± 0.4787 (1:3,P < 0.05) and 3.556 ± 0.2892 (1:5,P < 0.01). The p62 protein level was also increased. After transfection with p62 siRNA, the mRNA and protein levels of p62 were significantly decreased in BMDMs (P < 0.05). After 0.5, 1 and 2 hours of co-culture of BMDMs withC. albicans, flow cytometry showed that the phagocytosis rates ofC. albicans by BMDMs were significantly lower in the p62 siRNA group than in the NC group (39.70 ± 1.69%vs. 55.23 ± 0.72%, 46.70 ± 0.89%vs. 60.80 ± 1.78%, 51.90 ± 0.98%vs. 64.43 ± 2.0%, respectively, allP < 0.05). Consistent results were seen in the production of ROS (4269 ± 392.6vs. 13426 ± 1859.7, 4967 ± 721.2vs. 13687 ± 2611.2, 7647 ± 1950.0vs. 17719 ± 1814.2, respectively, allP < 0.05). The ROS levels were higher in BMDMs of the NC group than in BMDMs transfected with p62 siRNA at 0.5, 1, and 2 hours after treatment withC. albicans. BMDMs was co-cultured withC. albicans for 4 and 12 hours, the mRNA levels of interleukin-1β and interleukin-18 in NCs were also higher than p62 siRNA group, interleukin-1β: (6.14 ± 1.63vs. 12.12 ± 0.54, 8.81 ± 0.86vs. 26.2 ± 4.67, respectively, allP < 0.05), IL-18: (0.38 ± 0.02vs. 0.97 ± 0.06, 0.44 ± 0.02vs. 2.23 ± 0.46, respectively, allP < 0.05).Conclusion: p62 plays an important role in the process of phagocytosis in BMDMs challenged byC. albicans through ROS production and expression of proinflammatory cytokines.
基金supported by the NHMRC-ARC Dementia Research Development Fellowship Grant(AP1102977)an Australian Government Research Training Program(RTS)Scholarship。
文摘Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditionally mutations manifesting in the ubiquitin-associated domain of p62 were associated with Paget’s disease of bone,mutations affecting all functional domains of p62 have now been identified in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.p62 is a multifunctional protein that facilitates protein degradation through autophagy and the ubiquitin-proteasome system,and also regulates cell survival via the Nrf2 antioxidant response pathway,the nuclear factor-kappa B signaling pathway and apoptosis.Dysfunction in these signaling and protein degradation pathways have been observed in amyotrophic lateral sclerosis and frontotemporal lobar degeneration,and mutations that affect the role of p62 in these pathways may contribute to disease pathogenesis.In this review we discuss the role of p62 in these pathways,the effects of p62 mutations and the effect of mutations in the p62 modulator TANK-binding kinase 1,in relation to amyotrophic lateral sclerosis-frontotemporal lobar degeneration pathogenesis.
基金supported by grants from the National Natural Science Foundation of China to R.J.(31529004,31671422,and 31601112)the 111 Project(D18010)+1 种基金the Local Innovative and Research Teams Project of Guangdong Perl River Talents Program(2017BT01S155)the China Postdoctoral Science Foundation to C.W.(2018M640767)
文摘Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.
