BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR typ...BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.展开更多
目的利用基于荧光共振能量转移(Fluorescence resonance energy transfer,FRET)技术的检测PKC激活或PKC-delta激活的报告分子来确定PTH是否可以通过PLC非依赖途径激活PKC和PKC-delta。方法将表达PKC激活报告分子(CKAR)的质粒和表达PKC-d...目的利用基于荧光共振能量转移(Fluorescence resonance energy transfer,FRET)技术的检测PKC激活或PKC-delta激活的报告分子来确定PTH是否可以通过PLC非依赖途径激活PKC和PKC-delta。方法将表达PKC激活报告分子(CKAR)的质粒和表达PKC-delta激活报告分子的质粒转染HEK293细胞,培养72h后通过共聚焦显微镜检测FRET的改变,并以此判断佛波酯(TPA)是否激活PKC和PKC-delta。将表达甲状旁腺素1型受体(PTHR1)的质粒与CKAR质粒或PKC-delta质粒共转染HEK293细胞,培养72h后通过共聚焦显微镜检测FRET的改变,并判断PTH(1-34)、G1R19(1-34)和0.1%的三氟乙酸(TFA)对PKC和PKC-delta的作用。结果在转染CKAR质粒或PKC-delta质粒的HEK293细胞,TPA均使青色荧光与黄色荧光的强度之比(C/Y)增加。在共转染PTH1R质粒与CKAR质粒或PKC-delta质粒的HEK293细胞,PTH(1-34)和G1R19(1-34)均增加了C/Y的值,而0.1%TFA未引起C/Y的改变。结论 PTH与PTHR1结合后通过PLC非依赖途径激活PKC/PKC-delta。展开更多
目的通过观察不同血液净化方式的联合应用对血液透析患者血清中骨代谢指标及骨质疏松患病率的影响,来评价血液透析方式的不同组合对血液透析患者矿物质和骨异常(mineral and bone disorder,MBD)的影响。方法维持性血液透析患者90例,随...目的通过观察不同血液净化方式的联合应用对血液透析患者血清中骨代谢指标及骨质疏松患病率的影响,来评价血液透析方式的不同组合对血液透析患者矿物质和骨异常(mineral and bone disorder,MBD)的影响。方法维持性血液透析患者90例,随机分为血液透析组(对照组或HD组)、血液透析加血液透析滤过组(HD+HDF组)、血液透析加血液灌流组(HD+HP组),每组30例,观察6个月,比较组内及组间6个月前后血钙、血磷、全段甲状旁腺激素(intact parathyroid hormone,iPTH)、成纤维生长因子23(fibroblast growth factor23,FGF-23)、β-I型胶原羧基末端肽(β-Type I collagen carboxy-terminal peptide,β-CTX)、I型前胶原氨基末端肽(Type I procollagen amino-terminal peptide,PINP)、骨质疏松患病率等指标的变化来评价不同透析方式的组合对血液透析患者MBD的影响。结果组内自身前后比较:HD组血磷[(2.21±0.55)mmol/l vs.(2.64±1.04)mmol/l,t=2.047、P=0.049]、iPTH[(427.7±44.00)pg/ml vs.(452.1±43.00)pg/ml,t=2.140,P=0.038]变化有统计学差异;HD+HDF组的iPTH下降有统计学差异[(465.3±43.02)pg/ml vs.(431.0±37.39)pg/ml,t=3.298,P=0.007];HD+HP组的iPTH[(457.4±60.01)pg/mlvs.(389.1±29.89)pg/ml,t=5.598,P=0.001)、β-CTX[(2.73±1.16)ng/ml vs.(2.13±1.51)ng/ml,t=2.142,P=0.045];PINP[(157.92±31.16)ng/mlvs.(140.76±36.13)ng/ml,t=2.106,P=0.047];FGF-23[(461.16±101.69)ng/ml vs.(397.30±63.18)ng/ml,t=2.922,P=0.011]下降均有统计学意义。组间比较6个月后,HD+HDF组PINP为(147.33±40.72)ng/ml,HD组PINP为(165.32±43.11)ng/ml,二者比较有统计学差异(t=1.969,P=0.048);HD+HDF组FGF-23(465.38±101.36)ng/ml,HD组FGF-23(403.56±96.81)ng/ml,二者比较HD+HDF组更低,有统计学差异(t=2.415,P=0.019);组间比较6个月后,HD+HP组与HD组比较,血磷[(2.02±0.81)mmol/L vs.(2.64±1.04)mmol/L,t=3.221,P=0.003]、iPTH[(389.1±29.89)pg/ml vs.(452.1±43.0)pg/ml,t=6.661,P=0.005]、β-CTX[(2.13±1.51)ng/ml vs.(2.95±1.28)ng/ml,t=2.278,P=0.031]、PINP[(140.76±36.13)ng/ml vs.(165.32±43.11)ng/ml,t=2.339,P=0.028]、FGF-23[(397.30±63.18)ng/ml vs.(465.38±101.36)ng/ml,t=3.114,P=0.003],上述指标HD+HP组较HD组更低,均有统计学意义;组间比较6个月后,HD+HP组与HD+HDF组比较iPTH分别为(389.1±29.89)ng/ml、(431.0±37.39)ng/ml,HD+HP组更低,有统计学意义(t=7.303,P=0.000)。骨质疏松患病率:对照组观察期后骨质疏松和骨量低下的病例数增加,但无统计学意义;HD+HDF组、HD+HP组骨质疏松的病例数有减少,亦无统计学意义(P>0.05)。结论不同的血液透析方式的联合使用对维持性血液透析患者MBD影响不同;血液透析联合血液透析滤过或血液灌流的治疗方式较单纯血液透析有利MBD的改善;血液透析联合血液灌流在骨代谢指标的改善方面更具优势。展开更多
OBJECTIVE: To investigate whether the down-regulation of renal parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor messenger ribonucleic acid (mRNA) expression is a general phenomenon in patie...OBJECTIVE: To investigate whether the down-regulation of renal parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor messenger ribonucleic acid (mRNA) expression is a general phenomenon in patients with different stages of renal disease, besides chronic renal failure. METHODS: Twenty-five patients were divided into the following groups: (1) chronic glomerulonephritis with normal renal function (GNN) (2) chronic glomerulonephritis with moderate renal insufficiency (GNI) (3) severe chronic renal failure undergoing maintenance dialysis (CRF) (4) acute renal failure during oliguric phase (ARF) (5) normal control without renal suffering (NC). Using relatively quantitative reverse transcription/polymerase chain reaction (RT/PCR) method, we investigated PTH/PTHrP receptor mRNA expression in renal specimens obtained through biopsy or operation. The levels of plasma C-terminal PTH, serum phosphorus and calcium were also observed at the same time. RESULTS: Plasma C-terminal PTH levels in GNI, CRF and ARF patients were 1.90, 9.73 and 8.63 times higher than those in NC. However, the difference between GNN and NC was insignificant. CRF and ARF patients also presented obviously elevated serum phosphorus (1.61, 1.86 vs 1.14 mmol/L) and reduced serum calcium (1.82, 1.71 vs 2.26 mmol/L) compared with that in the control. These two parameters for GNN and GNI patients were normal. The levels of PTH/PTHrP receptor mRNA (corrected by beta-actin mRNA) in the kidney of GNN, GNI, CRF and ARF patients was markedly decreased by up to 35.7%, 68.5%, 77.9% and 92.2%, respectively. CONCLUSIONS: The down-regulation of renal PTH/PTHrP receptor mRNA occurs much earlier than the changes of renal function, plasma PTH, serum phosphorus and calcium in the course of human renal disease.展开更多
基金Supported by the Agencia Nacional de Promoción Científica y TecnológicaNo. PICT-2013-1441+8 种基金Consejo Nacional de Investigaciones Científicas y TécnicasNo. PIP11220150100350Instituto Nacional del Cáncer Asistencia Financiera ⅡRESOL 493/14, No. 2002-4395-14-1Instituto Nacional del Cáncer Asistencia Financiera Ⅲ-2016-2017, RESOL-2016-1006-E-APN-MSNo. 2002-3862-16-1 CANCERUniversidad Nacional del SurNo. PGI:24/B230 and No. PGI:24/B303Fundación Alberto J. Roemmers of Argentina
文摘BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.
文摘目的利用基于荧光共振能量转移(Fluorescence resonance energy transfer,FRET)技术的检测PKC激活或PKC-delta激活的报告分子来确定PTH是否可以通过PLC非依赖途径激活PKC和PKC-delta。方法将表达PKC激活报告分子(CKAR)的质粒和表达PKC-delta激活报告分子的质粒转染HEK293细胞,培养72h后通过共聚焦显微镜检测FRET的改变,并以此判断佛波酯(TPA)是否激活PKC和PKC-delta。将表达甲状旁腺素1型受体(PTHR1)的质粒与CKAR质粒或PKC-delta质粒共转染HEK293细胞,培养72h后通过共聚焦显微镜检测FRET的改变,并判断PTH(1-34)、G1R19(1-34)和0.1%的三氟乙酸(TFA)对PKC和PKC-delta的作用。结果在转染CKAR质粒或PKC-delta质粒的HEK293细胞,TPA均使青色荧光与黄色荧光的强度之比(C/Y)增加。在共转染PTH1R质粒与CKAR质粒或PKC-delta质粒的HEK293细胞,PTH(1-34)和G1R19(1-34)均增加了C/Y的值,而0.1%TFA未引起C/Y的改变。结论 PTH与PTHR1结合后通过PLC非依赖途径激活PKC/PKC-delta。
文摘OBJECTIVE: To investigate whether the down-regulation of renal parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor messenger ribonucleic acid (mRNA) expression is a general phenomenon in patients with different stages of renal disease, besides chronic renal failure. METHODS: Twenty-five patients were divided into the following groups: (1) chronic glomerulonephritis with normal renal function (GNN) (2) chronic glomerulonephritis with moderate renal insufficiency (GNI) (3) severe chronic renal failure undergoing maintenance dialysis (CRF) (4) acute renal failure during oliguric phase (ARF) (5) normal control without renal suffering (NC). Using relatively quantitative reverse transcription/polymerase chain reaction (RT/PCR) method, we investigated PTH/PTHrP receptor mRNA expression in renal specimens obtained through biopsy or operation. The levels of plasma C-terminal PTH, serum phosphorus and calcium were also observed at the same time. RESULTS: Plasma C-terminal PTH levels in GNI, CRF and ARF patients were 1.90, 9.73 and 8.63 times higher than those in NC. However, the difference between GNN and NC was insignificant. CRF and ARF patients also presented obviously elevated serum phosphorus (1.61, 1.86 vs 1.14 mmol/L) and reduced serum calcium (1.82, 1.71 vs 2.26 mmol/L) compared with that in the control. These two parameters for GNN and GNI patients were normal. The levels of PTH/PTHrP receptor mRNA (corrected by beta-actin mRNA) in the kidney of GNN, GNI, CRF and ARF patients was markedly decreased by up to 35.7%, 68.5%, 77.9% and 92.2%, respectively. CONCLUSIONS: The down-regulation of renal PTH/PTHrP receptor mRNA occurs much earlier than the changes of renal function, plasma PTH, serum phosphorus and calcium in the course of human renal disease.
