Cancers are characterized by deregulation of multiple signaling pathways and thus monotherapies are hardly effective. Neuroblastoma, which often occurs in adrenal glands, is the most common childhood malignancy. Malig...Cancers are characterized by deregulation of multiple signaling pathways and thus monotherapies are hardly effective. Neuroblastoma, which often occurs in adrenal glands, is the most common childhood malignancy. Malignant neuroblastoma resists traditional treatments and further studies are needed for effective therapeutic interventions. We evaluated synergistic efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and genistein (GST) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells in culture and activation of multiple pathways for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice. Combination of 4-HPR and GST synergistically reduced cell viability, caused subG1 accumulation, increased caspase-3 activity for apoptosis in vitro and reduced tumor growth in vivo. Western blotting indicated that combination therapy down regulated Id2 to induce differentiation, increased pro-apoptotic Bax and decreased anti-apoptotic Bcl-2 leading to an increase in Bax:Bcl-2 ratio, increased mitochondrial Bax level, caused mitochondrial release of Smac/Diablo, down regulation of the baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3, and activation of calpain and caspase-3 in SH-SY5Y xenografts. Accumulation of apoptosis-inducing-factor (AIF) in cytosol and increase in caspase-4 activation suggested involvement of mitochondrial pathway and endoplasmic reticulum (ER) stress, respectively, for apoptosis in SH-SY5Y xenografts. In situ immunofluorescent labelings of SH-SY5Y and SK-N-BE2 xenograft sections showed overexpression of calpain, caspase-12, and caspase-3, and AIF, suggesting induction of mitochondrial caspase-dependent and caspase-independent pathways for apoptosis. Collectively, synergistic effects of 4-HPR and GST induced mitochondrial pathways and also ER stress for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice.展开更多
目的探讨载N-（4-羟基苯基）维甲酰胺（4HPR）及4HPR脂质体（4HPR-L）与4HPR脂质微泡（4HPR-LM）联合超声对人瘢痕疙瘩成纤维细胞（Fb）增殖、凋亡及细胞周期的影响。方法（1）采用水化超声法制备4HPR-L及4HPR-LM，采用高效液相色谱法...目的探讨载N-（4-羟基苯基）维甲酰胺（4HPR）及4HPR脂质体（4HPR-L）与4HPR脂质微泡（4HPR-LM）联合超声对人瘢痕疙瘩成纤维细胞（Fb）增殖、凋亡及细胞周期的影响。方法（1）采用水化超声法制备4HPR-L及4HPR-LM，采用高效液相色谱法、动态光散射法、透射电镜对4HPR-L外观形态、粒径分布、Zeta电势、载药浓度、包封率、载药量进行考察。（2）取人瘢痕疙瘩Fb，采用随机数字表法（分组方法下同）分为13组，每组6孔。对照组细胞不给予任何处理，0.5 W 30 s组、0.5 W 60 s组、0.5 W 120 s组、0.7 W 30 s组、0.7 W 60 s组、0.7 W 120 s组、1.0 W 30 s组、1.0 W 60 s组、1.0 W 120 s组、1.5 W 30 s组、1.5 W 60 s组、1.5 W 120 s组12个超声组细胞分别给予对应参数超声处理。常规培养24 h后，酶标仪测定细胞活力。另取人瘢痕疙瘩Fb，分为5组，每组6孔。对照组细胞不给予任何处理，1、10、20、50 μg/mL空白脂质微泡组细胞给予对应质量浓度的空白脂质微泡处理，常规培养24 h后同前测定细胞活力。另取人瘢痕疙瘩Fb，分为6组，每组12孔。对照组细胞不给予任何处理，1、10、20、50、100 μg/mL 4HPR-L组细胞分别加入含对应质量浓度4HPR的4HPR-L处理，常规培养24、48 h后每组各取6孔同前测定细胞活力。另取人瘢痕疙瘩Fb，分为4组，每组6孔。对照组细胞不给予任何处理，4HPR组、4HPR-L组和4HPR-LM＋超声组细胞分别加入4HPR、4HPR-L、4HPR-LM（4HPR质量浓度均为20 μg/mL）处理，4HPR-LM＋超声组细胞给药后立即给予0.5 W 60 s超声处理。常规培养24 h后同前测定细胞活力。（3）取人瘢痕疙瘩Fb，分为对照组、4HPR组、4HPR-L组和4HPR-LM＋超声组，每组3孔，各组细胞处理同前。常规培养24 h后流式细胞仪检测细胞凋亡情况。（4）取人瘢痕疙瘩Fb，同（3）分组及处理，常规培养24 h后流式细胞仪检测细胞周期分布情况。对数�展开更多
Background:Previously,dihydroceramide(d18:0/24:0)(dhCer(d18:0/24:0))was reported to be a potential biomarker for acute-onchronic liver failure(ACLF)prognosis.In this study,we further explored the role of dhCer(d18:0/2...Background:Previously,dihydroceramide(d18:0/24:0)(dhCer(d18:0/24:0))was reported to be a potential biomarker for acute-onchronic liver failure(ACLF)prognosis.In this study,we further explored the role of dhCer(d18:0/24:0)in the progression of ACLF to validate the biomarker using ACLF rat model.Methods:ACLF rats were sacrificed at 4 and 8 h post-D-galactosamine(D-gal)/lipopolysaccharide(LPS)administration to investigate the liver biochemical markers,prothrombin time and liver histopathology.Change in dhCer and other sphingolipids levels were investigated by high-performance liquid chromatography coupled to tandem mass spectrometry(HPLC-MS/MS).Rats were treated with N-(4-hydroxyphenyl)retinamide(4-HPR)to examine the mortality rate and its role in improving ACLF.Results:LPS/D-gal administration resulted in significant elevation in alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels.Prothrombin time was prolonged and histopathological examination showed abnormality.HPLC-MS/MS results showed total dhCer levels in ACLF group(64.10±8.90 pmol/100 mL,64.22±6.78 pmol/100 mL for 4 and 8 h,respectively)were decreased significantly compared with control group(121.61±23.09 pmol/100 mL)(P<0.05).In particular,dhCer(d18:0/24:0),dhCer(d18:0/20:0),and dhCer(d18:0/22:0)levels were decreased.Treatment with 4-HPR significantly increased the levels of dhCers,including dhCer(d18:0/24:0)compared with ACLF group,for the level of dhCer(d18:0/24:0)in 4-HPR group was 20.10±8.60 pmol/100 mL and the level of dhCer(d18:0/24:0)in ACLF group was 9.74±2.99 pmol/100 mL(P<0.05).This was associated with reduced mortality rate and prolonged survival time.The ALT and AST in 4-HPR group were significantly decreased compared with ACLF group.The prothrombin time of 4-HPR group(41.49 s)was significantly lower than the prothrombin time of ACLF group(57.96 s)(P<0.05).4-HPR also decreased plasma ammonia levels slightly,as the plasma ammonia levels in 4-HPR group and ACLF group were 207.37±60.43,209.15±60.43 mmol/L,respectively.Furthe展开更多
文摘Cancers are characterized by deregulation of multiple signaling pathways and thus monotherapies are hardly effective. Neuroblastoma, which often occurs in adrenal glands, is the most common childhood malignancy. Malignant neuroblastoma resists traditional treatments and further studies are needed for effective therapeutic interventions. We evaluated synergistic efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and genistein (GST) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells in culture and activation of multiple pathways for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice. Combination of 4-HPR and GST synergistically reduced cell viability, caused subG1 accumulation, increased caspase-3 activity for apoptosis in vitro and reduced tumor growth in vivo. Western blotting indicated that combination therapy down regulated Id2 to induce differentiation, increased pro-apoptotic Bax and decreased anti-apoptotic Bcl-2 leading to an increase in Bax:Bcl-2 ratio, increased mitochondrial Bax level, caused mitochondrial release of Smac/Diablo, down regulation of the baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3, and activation of calpain and caspase-3 in SH-SY5Y xenografts. Accumulation of apoptosis-inducing-factor (AIF) in cytosol and increase in caspase-4 activation suggested involvement of mitochondrial pathway and endoplasmic reticulum (ER) stress, respectively, for apoptosis in SH-SY5Y xenografts. In situ immunofluorescent labelings of SH-SY5Y and SK-N-BE2 xenograft sections showed overexpression of calpain, caspase-12, and caspase-3, and AIF, suggesting induction of mitochondrial caspase-dependent and caspase-independent pathways for apoptosis. Collectively, synergistic effects of 4-HPR and GST induced mitochondrial pathways and also ER stress for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice.
文摘目的探讨载N-（4-羟基苯基）维甲酰胺（4HPR）及4HPR脂质体（4HPR-L）与4HPR脂质微泡（4HPR-LM）联合超声对人瘢痕疙瘩成纤维细胞（Fb）增殖、凋亡及细胞周期的影响。方法（1）采用水化超声法制备4HPR-L及4HPR-LM，采用高效液相色谱法、动态光散射法、透射电镜对4HPR-L外观形态、粒径分布、Zeta电势、载药浓度、包封率、载药量进行考察。（2）取人瘢痕疙瘩Fb，采用随机数字表法（分组方法下同）分为13组，每组6孔。对照组细胞不给予任何处理，0.5 W 30 s组、0.5 W 60 s组、0.5 W 120 s组、0.7 W 30 s组、0.7 W 60 s组、0.7 W 120 s组、1.0 W 30 s组、1.0 W 60 s组、1.0 W 120 s组、1.5 W 30 s组、1.5 W 60 s组、1.5 W 120 s组12个超声组细胞分别给予对应参数超声处理。常规培养24 h后，酶标仪测定细胞活力。另取人瘢痕疙瘩Fb，分为5组，每组6孔。对照组细胞不给予任何处理，1、10、20、50 μg/mL空白脂质微泡组细胞给予对应质量浓度的空白脂质微泡处理，常规培养24 h后同前测定细胞活力。另取人瘢痕疙瘩Fb，分为6组，每组12孔。对照组细胞不给予任何处理，1、10、20、50、100 μg/mL 4HPR-L组细胞分别加入含对应质量浓度4HPR的4HPR-L处理，常规培养24、48 h后每组各取6孔同前测定细胞活力。另取人瘢痕疙瘩Fb，分为4组，每组6孔。对照组细胞不给予任何处理，4HPR组、4HPR-L组和4HPR-LM＋超声组细胞分别加入4HPR、4HPR-L、4HPR-LM（4HPR质量浓度均为20 μg/mL）处理，4HPR-LM＋超声组细胞给药后立即给予0.5 W 60 s超声处理。常规培养24 h后同前测定细胞活力。（3）取人瘢痕疙瘩Fb，分为对照组、4HPR组、4HPR-L组和4HPR-LM＋超声组，每组3孔，各组细胞处理同前。常规培养24 h后流式细胞仪检测细胞凋亡情况。（4）取人瘢痕疙瘩Fb，同（3）分组及处理，常规培养24 h后流式细胞仪检测细胞周期分布情况。对数�
基金This work was supported by the National Natural Science Foundation of China(No.81573487)the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(No.2017-12M-1-013)the Drug Innovation Major Project(No.2018ZX09711001-003-011).
文摘Background:Previously,dihydroceramide(d18:0/24:0)(dhCer(d18:0/24:0))was reported to be a potential biomarker for acute-onchronic liver failure(ACLF)prognosis.In this study,we further explored the role of dhCer(d18:0/24:0)in the progression of ACLF to validate the biomarker using ACLF rat model.Methods:ACLF rats were sacrificed at 4 and 8 h post-D-galactosamine(D-gal)/lipopolysaccharide(LPS)administration to investigate the liver biochemical markers,prothrombin time and liver histopathology.Change in dhCer and other sphingolipids levels were investigated by high-performance liquid chromatography coupled to tandem mass spectrometry(HPLC-MS/MS).Rats were treated with N-(4-hydroxyphenyl)retinamide(4-HPR)to examine the mortality rate and its role in improving ACLF.Results:LPS/D-gal administration resulted in significant elevation in alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels.Prothrombin time was prolonged and histopathological examination showed abnormality.HPLC-MS/MS results showed total dhCer levels in ACLF group(64.10±8.90 pmol/100 mL,64.22±6.78 pmol/100 mL for 4 and 8 h,respectively)were decreased significantly compared with control group(121.61±23.09 pmol/100 mL)(P<0.05).In particular,dhCer(d18:0/24:0),dhCer(d18:0/20:0),and dhCer(d18:0/22:0)levels were decreased.Treatment with 4-HPR significantly increased the levels of dhCers,including dhCer(d18:0/24:0)compared with ACLF group,for the level of dhCer(d18:0/24:0)in 4-HPR group was 20.10±8.60 pmol/100 mL and the level of dhCer(d18:0/24:0)in ACLF group was 9.74±2.99 pmol/100 mL(P<0.05).This was associated with reduced mortality rate and prolonged survival time.The ALT and AST in 4-HPR group were significantly decreased compared with ACLF group.The prothrombin time of 4-HPR group(41.49 s)was significantly lower than the prothrombin time of ACLF group(57.96 s)(P<0.05).4-HPR also decreased plasma ammonia levels slightly,as the plasma ammonia levels in 4-HPR group and ACLF group were 207.37±60.43,209.15±60.43 mmol/L,respectively.Furthe
