Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal mus...Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.展开更多
目的:研究内质网应激介导的PI3K/Akt抑制剂对肝癌细胞MEK/ERK途径的影响。方法:采用PI3K抑制剂LY294002/激活型突变载体myr-Akt分别阻断/激活内质网应激介导的Akt和ERK活化,并利用Western blot ting技术分析内质网应激条件下PI3K/Akt和M...目的:研究内质网应激介导的PI3K/Akt抑制剂对肝癌细胞MEK/ERK途径的影响。方法:采用PI3K抑制剂LY294002/激活型突变载体myr-Akt分别阻断/激活内质网应激介导的Akt和ERK活化,并利用Western blot ting技术分析内质网应激条件下PI3K/Akt和MEK/ERK途径间的关系。结果:阻断PI3K/Akt促进内质网应激介导的MEK/ERK活化,过度激活PI3K/Akt则抑制内质网应激介导的MEK/ERK活化。结论:PI3K/Akt和MEK/ERK信号途径间在内质网应激肝癌细胞中可能存在信号交流。展开更多
BACKGROUND Qingjie Fuzheng granules(QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, includi...BACKGROUND Qingjie Fuzheng granules(QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer(CRC).However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs' anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways.AIM To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8.METHOD First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase(LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21,Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3 K/AKT and ERK signaling pathwayswas examined using the western blot assay to investigate the possible mechanism.RESULTS MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by(6.90% ± 1.03%)–(59.70% ± 1.51%)(HCT-116; P < 0.05)and(5.56% ± 4.52%)–(49.44% ± 2.47%)(HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002(HCT-116; P < 0.01) and from 0.56 ±0.054 to 0.81 ± 0.044(HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells.QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3 K, AKT and ERK.CONCLUSION These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3 K/AKT and ERK signaling pathways.展开更多
基金supported by the National Natural Science Foundation of China,Nos.82171172(to RZ)and 81771366(to RZ)Fundamental Research Funds for the Central Universities of Central South University,Nos.2021zzts1095(to SZ)and 2022zzts0832(to HY)。
文摘Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.
文摘目的:研究内质网应激介导的PI3K/Akt抑制剂对肝癌细胞MEK/ERK途径的影响。方法:采用PI3K抑制剂LY294002/激活型突变载体myr-Akt分别阻断/激活内质网应激介导的Akt和ERK活化,并利用Western blot ting技术分析内质网应激条件下PI3K/Akt和MEK/ERK途径间的关系。结果:阻断PI3K/Akt促进内质网应激介导的MEK/ERK活化,过度激活PI3K/Akt则抑制内质网应激介导的MEK/ERK活化。结论:PI3K/Akt和MEK/ERK信号途径间在内质网应激肝癌细胞中可能存在信号交流。
基金Supported by Project Funding of the Scientific Research Foundation of Traditional Chinese Medicine of Fujian Provincial Health and Family Planning Commission,No.2017FJZYZY203the Training of Young and Middle-Aged Backbone Personnel of Fujian Provincial Health and Family Planning Commission,No.2016-ZQN-67/
文摘BACKGROUND Qingjie Fuzheng granules(QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer(CRC).However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs' anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways.AIM To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8.METHOD First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase(LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21,Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3 K/AKT and ERK signaling pathwayswas examined using the western blot assay to investigate the possible mechanism.RESULTS MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by(6.90% ± 1.03%)–(59.70% ± 1.51%)(HCT-116; P < 0.05)and(5.56% ± 4.52%)–(49.44% ± 2.47%)(HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002(HCT-116; P < 0.01) and from 0.56 ±0.054 to 0.81 ± 0.044(HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells.QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3 K, AKT and ERK.CONCLUSION These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3 K/AKT and ERK signaling pathways.
