Objective: α-ketoglutarate(α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α(HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of co...Objective: α-ketoglutarate(α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α(HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of collagen prolyl 4-hydroxylase in breast cancer cells stabilizes the expression of HIF-1α by depleting α-KG levels. We hypothesized that mitochondrial malic enzyme 2(ME2) might also affect HIF-1α expression via modulating α-KG levels in breast cancer cells.Methods: We evaluated ME2 protein expression in 100 breast cancer patients using immunohistochemistry and correlated with clinicopathological indicators. The effect of ME2 knockout on cancer metastasis was evaluated using an orthotopic breast cancer model. The effect of ME2 knockout or knockdown on the levels of α-KG and HIF-1α proteins in breast cancer cell lines was determined both in vitro and in vivo.Results: ME2 was found to be upregulated in the human breast cancerous tissues compared with the matched precancerous tissues(P<0.001). The elevated expression of ME2 was associated with a poor prognosis(P=0.019).ME2 upregulation was also related to lymph node metastasis(P=0.016), pathological staging(P=0.033), and vascular cancer embolus(P=0.014). Also, ME2 knockout significantly inhibited lung metastasis in vivo. In the tumors formed by ME2 knockout cells, the levels of α-KG were significantly increased and collagen hydroxylation level did not change significantly but HIF-1α protein expression was significantly decreased, compared to the control samples. In cell culture, cells with ME2 knockout or knockdown demonstrated significantly higher α-KG levels but significantly lower HIF-1α protein expression than control cells under hypoxia. Exogenous malate and α-KG exerted similar effect on HIF-1α in breast cancer cells to ME2 knockout or knockdown. Additionally,treatment with malate significantly decreased 4 T1 breast cancer lung metastasis. ME2 expression was associated with HIF-1α levels in human breast cancer samples(P=0.008).Conclusions: Our results provide evidence that upregulation of ME2 is associated with a poor prognosis of breast cancer patients and propose a mechanistic understanding of a link between ME2 and breast cancer metastasis.展开更多
Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation ...Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.展开更多
A large amount of nicotinamide adenine dinucleotide phosphate(NADPH) is required for fatty acid synthesis and maintenance of the redox state in cancer cells.Malic enzyme 1(ME1)-dependent NADPH production is one of the...A large amount of nicotinamide adenine dinucleotide phosphate(NADPH) is required for fatty acid synthesis and maintenance of the redox state in cancer cells.Malic enzyme 1(ME1)-dependent NADPH production is one of the three pathways that contribute to the formation of the cytosolic NADPH pool.ME1 is generally considered to be overexpressed in cancer cells to meet the high demand for increased de novo fatty acid synthesis.In the present study,we found that glucose induced higher ME1 activity and that repressing ME1 had a profound impact on glucose metabolism of nasopharyngeal carcinoma(NPC) cells.High incorporation of glucose and an enhancement of the pentose phosphate pathway were observed in ME1-repressed cells.However,there were no obvious changes in the other two pathways for glucose metabolism:glycolysis and oxidative phosphorylation.Interestingly,NADPH was decreased under low-glucose condition in ME1-repressed cells relative to wild-type cells,whereas no significant difference was observed under high-glucose condition.ME1-repressed cells had significantly decreased tolerance to low-glucose condition.Moreover,NADPH produced by ME1 was not only important for fatty acid synthesis but also essential for maintenance of the intracellular redox state and the protection of cells from oxidative stress.Furthermore,diminished migration and invasion were observed in ME1-repressed cells due to a reduced level of Snail protein.Collectively,these results suggest an essential role for ME1 in the production of cytosolic NADPH and maintenance of migratory and invasive abilities of NPC cells.展开更多
In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentra...In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.展开更多
基金supported in part by the China Natural Sciences Foundation projects (No. 81772947)。
文摘Objective: α-ketoglutarate(α-KG) is the substrate to hydroxylate collagen and hypoxia-inducible factor-1α(HIF-1α), which are important for cancer metastasis. Previous studies have shown that the upregulation of collagen prolyl 4-hydroxylase in breast cancer cells stabilizes the expression of HIF-1α by depleting α-KG levels. We hypothesized that mitochondrial malic enzyme 2(ME2) might also affect HIF-1α expression via modulating α-KG levels in breast cancer cells.Methods: We evaluated ME2 protein expression in 100 breast cancer patients using immunohistochemistry and correlated with clinicopathological indicators. The effect of ME2 knockout on cancer metastasis was evaluated using an orthotopic breast cancer model. The effect of ME2 knockout or knockdown on the levels of α-KG and HIF-1α proteins in breast cancer cell lines was determined both in vitro and in vivo.Results: ME2 was found to be upregulated in the human breast cancerous tissues compared with the matched precancerous tissues(P<0.001). The elevated expression of ME2 was associated with a poor prognosis(P=0.019).ME2 upregulation was also related to lymph node metastasis(P=0.016), pathological staging(P=0.033), and vascular cancer embolus(P=0.014). Also, ME2 knockout significantly inhibited lung metastasis in vivo. In the tumors formed by ME2 knockout cells, the levels of α-KG were significantly increased and collagen hydroxylation level did not change significantly but HIF-1α protein expression was significantly decreased, compared to the control samples. In cell culture, cells with ME2 knockout or knockdown demonstrated significantly higher α-KG levels but significantly lower HIF-1α protein expression than control cells under hypoxia. Exogenous malate and α-KG exerted similar effect on HIF-1α in breast cancer cells to ME2 knockout or knockdown. Additionally,treatment with malate significantly decreased 4 T1 breast cancer lung metastasis. ME2 expression was associated with HIF-1α levels in human breast cancer samples(P=0.008).Conclusions: Our results provide evidence that upregulation of ME2 is associated with a poor prognosis of breast cancer patients and propose a mechanistic understanding of a link between ME2 and breast cancer metastasis.
基金the National Natural Science Foundation of China,No.30900773the National University Basic Research Foundation of China,No.2010QZZD022
文摘Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.
基金supported by grants from the Major State Basic Research Program (973 Project) of China(No.2006CB910104 and 2010CB912201)the National High Technology Research and Development Program of China (863 Program) (No.20060102A4002)+1 种基金the State Key Program of National Natural Science Foundation of China (No.81030043)the Guangdong Province-National Natural Science Foundation of China Cooperation Program (No.u0732005)
文摘A large amount of nicotinamide adenine dinucleotide phosphate(NADPH) is required for fatty acid synthesis and maintenance of the redox state in cancer cells.Malic enzyme 1(ME1)-dependent NADPH production is one of the three pathways that contribute to the formation of the cytosolic NADPH pool.ME1 is generally considered to be overexpressed in cancer cells to meet the high demand for increased de novo fatty acid synthesis.In the present study,we found that glucose induced higher ME1 activity and that repressing ME1 had a profound impact on glucose metabolism of nasopharyngeal carcinoma(NPC) cells.High incorporation of glucose and an enhancement of the pentose phosphate pathway were observed in ME1-repressed cells.However,there were no obvious changes in the other two pathways for glucose metabolism:glycolysis and oxidative phosphorylation.Interestingly,NADPH was decreased under low-glucose condition in ME1-repressed cells relative to wild-type cells,whereas no significant difference was observed under high-glucose condition.ME1-repressed cells had significantly decreased tolerance to low-glucose condition.Moreover,NADPH produced by ME1 was not only important for fatty acid synthesis but also essential for maintenance of the intracellular redox state and the protection of cells from oxidative stress.Furthermore,diminished migration and invasion were observed in ME1-repressed cells due to a reduced level of Snail protein.Collectively,these results suggest an essential role for ME1 in the production of cytosolic NADPH and maintenance of migratory and invasive abilities of NPC cells.
基金This research was funded by Hebei Provincial Department of Science and Technology(21375501D)the Hebei Academy of Sciences(2019Q01).
文摘In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.
