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Regulation of autophagy via PERK-eIF2α effectively relieve the radiation myelitis induced by iodine-125.

Yang Z, Xu Y, Xu L, Maccauro G, Rossi B, Chen Y, Li H, Zhang J, Sun H, Yang Y, Xu D, Liu X - PLoS ONE (2013)

Bottom Line: The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells.The results were consistent with that by MTT and Annexin-FITC/PT staining.The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Kunming General Hospital of Chengdu Military Command, the Third Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, P. R. China ; Department of Orthopaedic Oncology, Agostino Gemelli Hospital, Catholic University of Rome, Largo Francesco Vito 1, Rome, Italy.

ABSTRACT
Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that (125)I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which (125)I radiation triggered autophagy in neural cells. We found that autophagy induced by (125)I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after (125)I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In animal model of banna pigs with radiation myelitis caused by (125)I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by (125)I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by (125)I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.

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The motor fuction changes between control and PERK-siRNA groups in each time point under 125I radiation.(A) Porcines transfected with RNAi for PERK and controls were radiated with 125I for 6 months. Protein levels were measured by immunoblot analysis using antibodies against LC3II and β-actin at each time point of 1 week, 1 month, 3 months and 6 months. (B) Annexin V-FITC/PI double staining analysis at different times. The lower left quadrant (low-fluorescence PI and FITC signals) contains normal viable cells and the lower right quadrant (low-fluorescence PI and high-fluorescence FITC signals) defines cells in the early stages of apoptosis. The two upper quadrants (high-fluorescence PI signal) define dead cells. The numbers indicate the percentages of the different populations of cells.
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pone-0076819-g007: The motor fuction changes between control and PERK-siRNA groups in each time point under 125I radiation.(A) Porcines transfected with RNAi for PERK and controls were radiated with 125I for 6 months. Protein levels were measured by immunoblot analysis using antibodies against LC3II and β-actin at each time point of 1 week, 1 month, 3 months and 6 months. (B) Annexin V-FITC/PI double staining analysis at different times. The lower left quadrant (low-fluorescence PI and FITC signals) contains normal viable cells and the lower right quadrant (low-fluorescence PI and high-fluorescence FITC signals) defines cells in the early stages of apoptosis. The two upper quadrants (high-fluorescence PI signal) define dead cells. The numbers indicate the percentages of the different populations of cells.

Mentions: In our previous studies, we have successfully established radiation myelitis animal model caused by 125I brachytherapy in banna pigs. One of the groups was successfully reduced PERK expression by intrathecal administration of the lentivirus vector (PERK-siRNA group), the other was control group with normal PERK expression that was by intrathecal administration of medium (Control group). We detected the expression of autophagy marker LC3II by western blot assay, and the results showed the PERK expression was significantly inhibited within 1 to 6 months after injection. Apoptosis and autophagy rates were also detected by FACS assay using Annexin V-FITC/PI double staining analysis. As demonstrated in Table 1 and Figure 7B, apoptosis rates were significantly higher than that in control group. Consistent with the results, we also observed more serious histological impairments and moving difficulty for banna mini-pigs in PERK-siRNA group.


Regulation of autophagy via PERK-eIF2α effectively relieve the radiation myelitis induced by iodine-125.

Yang Z, Xu Y, Xu L, Maccauro G, Rossi B, Chen Y, Li H, Zhang J, Sun H, Yang Y, Xu D, Liu X - PLoS ONE (2013)

The motor fuction changes between control and PERK-siRNA groups in each time point under 125I radiation.(A) Porcines transfected with RNAi for PERK and controls were radiated with 125I for 6 months. Protein levels were measured by immunoblot analysis using antibodies against LC3II and β-actin at each time point of 1 week, 1 month, 3 months and 6 months. (B) Annexin V-FITC/PI double staining analysis at different times. The lower left quadrant (low-fluorescence PI and FITC signals) contains normal viable cells and the lower right quadrant (low-fluorescence PI and high-fluorescence FITC signals) defines cells in the early stages of apoptosis. The two upper quadrants (high-fluorescence PI signal) define dead cells. The numbers indicate the percentages of the different populations of cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818370&req=5

pone-0076819-g007: The motor fuction changes between control and PERK-siRNA groups in each time point under 125I radiation.(A) Porcines transfected with RNAi for PERK and controls were radiated with 125I for 6 months. Protein levels were measured by immunoblot analysis using antibodies against LC3II and β-actin at each time point of 1 week, 1 month, 3 months and 6 months. (B) Annexin V-FITC/PI double staining analysis at different times. The lower left quadrant (low-fluorescence PI and FITC signals) contains normal viable cells and the lower right quadrant (low-fluorescence PI and high-fluorescence FITC signals) defines cells in the early stages of apoptosis. The two upper quadrants (high-fluorescence PI signal) define dead cells. The numbers indicate the percentages of the different populations of cells.
Mentions: In our previous studies, we have successfully established radiation myelitis animal model caused by 125I brachytherapy in banna pigs. One of the groups was successfully reduced PERK expression by intrathecal administration of the lentivirus vector (PERK-siRNA group), the other was control group with normal PERK expression that was by intrathecal administration of medium (Control group). We detected the expression of autophagy marker LC3II by western blot assay, and the results showed the PERK expression was significantly inhibited within 1 to 6 months after injection. Apoptosis and autophagy rates were also detected by FACS assay using Annexin V-FITC/PI double staining analysis. As demonstrated in Table 1 and Figure 7B, apoptosis rates were significantly higher than that in control group. Consistent with the results, we also observed more serious histological impairments and moving difficulty for banna mini-pigs in PERK-siRNA group.

Bottom Line: The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells.The results were consistent with that by MTT and Annexin-FITC/PT staining.The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Kunming General Hospital of Chengdu Military Command, the Third Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, P. R. China ; Department of Orthopaedic Oncology, Agostino Gemelli Hospital, Catholic University of Rome, Largo Francesco Vito 1, Rome, Italy.

ABSTRACT
Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that (125)I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which (125)I radiation triggered autophagy in neural cells. We found that autophagy induced by (125)I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after (125)I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In animal model of banna pigs with radiation myelitis caused by (125)I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by (125)I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by (125)I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.

Show MeSH
Related in: MedlinePlus