BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) a...BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma(HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHPassociated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIM To confirm the effect of XHP on HCC and the possible mechanisms involved.METHODS The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS). Cellbased experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP(0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay.Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction(RT-qPCR), respectively.Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway.Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTS The following 12 compounds were identified in XHP using high-resolution mass spectrometry:Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose-and timedependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract(0.625mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins(e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSION XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3.Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.展开更多
Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of ...Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer.In this work,we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer.Methods:The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform.The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform.The target genes of liver cancer were predicted using GeneCards database.The intersecting genes were then analyzed with Venn diagrams.Kyoto Encyclopedia of Genes and Genomes and Database for Annotation,Visualization,and Integrated Discovery were used to analyze the pathway.Then,cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills.The living dead cell staining method was used to observe the survival of cells.HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method,and then the mitochondrial damage was also detected by flow cytometry.The expression of target genes was detected by quantitative real-time polymerase chain reaction.Results:A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer‑related targets.We obtained 1,899 disease targets and 297 transcriptome targets from the database.Six drug-disease intersecting genes,CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3 were obtained.They are enrichment in apoptosis,PI3K-AKT signaling pathway,MAPK signaling pathway,pathways in cancer and p53 signaling pathway.Besides,it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group.And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment.Xihuang pill also induced the mitochondrial membrane potential damage.Compared with the control group,the expression level of CCNB1 and BIRC5 was induced,while the expression level of IGF2 was reduced after Xihuang pill treatment.Conclusion:Xihuang pill may act on six proteins(CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3)and cover multiple pathways to form a therapeutic network to treat liver cancer.展开更多
Objective：To investigate the antiproliferative and anti-metastasis effect of Xihuang Pill（西黄丸,XP） on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects.Methods：...Objective：To investigate the antiproliferative and anti-metastasis effect of Xihuang Pill（西黄丸,XP） on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects.Methods：Highly metastatic human colorectal cancer cell line LoVo was treated with low-,medium-,and highdose XP-containing serum（XP-L,XP-M,XP-H） groups for 48 h,cells intervened with no drug rat serum and PD98059[extracellular signal-regulated kinase（ERK） inhibitor]as negative and positive controls（NC and PC）groups.Cell proliferation assay was made using cell counting kit-8（CCK8）.The 8 μm pore-size transwell chamber and 4＇,6-diamidino-2-phenylindole（DAPI） staining were applied to examine the ability of invasion and migration of the cells.The protein expression of ERK1/2,zinc finger E-box-binding homeobox 1（ZEB1）,Scrib and lethal giant larvae homolog 2（Lgl2） was detected by Western blotting while the relative mRNA quantity of E-cadherin,N-cadherin,Occludin and junctional adhesion molecule-1（JAM1） was measured by realtime fluorescent quantitative polymerase chain reaction（RT-qPCR）.Results：XP induced a dose-dependent suppression on the proliferation of LoVo cells（P〈0.05 or P〈0.01）,with the inhibition rates varied from 27.30%to31.08%.Transwell assay showed that when preprocessed with PD98059 and XP-containing serum,the number of cells that passed the filter decreased significantly compared with that of NC group（P〈0.05 or P〈0.01）.Moreover,XP inhibited the protein expression of ERK1/2 and ZEB1（P〈0.05）;and up-regulated the protein expression of Scrib and Lgl2（P〈0.05）.The mRNA levels of E-cadherin,Occludin and JAM1 of the XP intervened groups and PC group markedly ascended（P〈0.05） while that of N-cadherin showed a descending tendency（P〉0.05）.Conclusion：XP intervention suppressed the ability of proliferation,invasion and migration of the LoVo cells.Regulating ZEB1-SCRIB Loop so as to recover epithelial phenotype and apical junctional complex might be one of the mechanisms by which XP produces the anti-metastasis effect.展开更多
OBJECTIVE： To investigate the anti-breast cancer （BC） effects and mechanisms of action of Xihuang pill （XHP） by conducting in vitro experiments on hu- man BC cell lines. METHODS： Two human BC cell lines （MCF-7 ...OBJECTIVE： To investigate the anti-breast cancer （BC） effects and mechanisms of action of Xihuang pill （XHP） by conducting in vitro experiments on hu- man BC cell lines. METHODS： Two human BC cell lines （MCF-7 and MDA- MB231） were cultured and treated with XHP. Cell viability was detected using the 3-（4, 5-Dimeth- ylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay. Flow cytometry was used to measure the cell cycle and apoptosis. The cell cycle was ana- lyzed with propidium iodide staining. Apoptosis was evaluated using the Annexin V-fluorescein iso- thiocyanate/propidium iodide method. Western blotting was used to analyze the expression of es- trogen receptor （ER）-α and ER-13.RESULTS： XHP had growth-inhibitory effects on MCF-7 and MDA-MB231 cells with a half-maximal inhibitory concentration （IC50） of 10.14 mg/mL （MCF-7） and 8.98 mg/mL （MDA-MB231）. Apoptosis was induced to some extent. Certain changes in the ER were caused. Upregulation of ER-a protein was found in MCF-7 cells. ER-β expression in MDA-MB231 cells was increased. Cell-cycle arrest was not observed in the two BC cell lines. ER-β ex- pression in MCF-7 cells was unchanged. No ER-a ex- pression was shown in MDA-MB231 cells. CONCLUSION： These data suggest that XHP can af- fect cell viability and cause apoptosis, but that the cell cycle is not blocked. XHP has a certain impact on ER expression, but its mechanisms of action of anti-13C effects may not be due to regulation of ER expression.展开更多
基金Supported by National Natural Science Foundation of China, No. U20A20408 and No. 82074450Natural Science Foundation of Hunan Province, No. 2020JJ4066+4 种基金Hunan Province"Domestic First-class Cultivation Discipline"Integrated Traditional Chinese and Western Medicine Open Fund Project, No. 2020ZXYJH34 and No. 2020ZXYJH35Hunan Graduate Scientific Research Innovation Project, No. QL20210173 and No. CX20210730Hunan Province Science and Technology Innovation Talents Plan College Students Science and Technology Innovation and Entrepreneurship Project, No. 2020RC1004Guangzhou Health Science and Technology Project, No. 20221A011102Hunan Traditional Chinese Medicine Scientific Research Project, No. 202101
文摘BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma(HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHPassociated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIM To confirm the effect of XHP on HCC and the possible mechanisms involved.METHODS The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS). Cellbased experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP(0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay.Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction(RT-qPCR), respectively.Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway.Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTS The following 12 compounds were identified in XHP using high-resolution mass spectrometry:Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose-and timedependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract(0.625mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins(e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSION XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3.Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.
文摘Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer.In this work,we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer.Methods:The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform.The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform.The target genes of liver cancer were predicted using GeneCards database.The intersecting genes were then analyzed with Venn diagrams.Kyoto Encyclopedia of Genes and Genomes and Database for Annotation,Visualization,and Integrated Discovery were used to analyze the pathway.Then,cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills.The living dead cell staining method was used to observe the survival of cells.HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method,and then the mitochondrial damage was also detected by flow cytometry.The expression of target genes was detected by quantitative real-time polymerase chain reaction.Results:A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer‑related targets.We obtained 1,899 disease targets and 297 transcriptome targets from the database.Six drug-disease intersecting genes,CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3 were obtained.They are enrichment in apoptosis,PI3K-AKT signaling pathway,MAPK signaling pathway,pathways in cancer and p53 signaling pathway.Besides,it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group.And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment.Xihuang pill also induced the mitochondrial membrane potential damage.Compared with the control group,the expression level of CCNB1 and BIRC5 was induced,while the expression level of IGF2 was reduced after Xihuang pill treatment.Conclusion:Xihuang pill may act on six proteins(CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3)and cover multiple pathways to form a therapeutic network to treat liver cancer.
基金Supported by the National Natural Science Foundation of China(No.81273636)
文摘Objective：To investigate the antiproliferative and anti-metastasis effect of Xihuang Pill（西黄丸,XP） on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects.Methods：Highly metastatic human colorectal cancer cell line LoVo was treated with low-,medium-,and highdose XP-containing serum（XP-L,XP-M,XP-H） groups for 48 h,cells intervened with no drug rat serum and PD98059[extracellular signal-regulated kinase（ERK） inhibitor]as negative and positive controls（NC and PC）groups.Cell proliferation assay was made using cell counting kit-8（CCK8）.The 8 μm pore-size transwell chamber and 4＇,6-diamidino-2-phenylindole（DAPI） staining were applied to examine the ability of invasion and migration of the cells.The protein expression of ERK1/2,zinc finger E-box-binding homeobox 1（ZEB1）,Scrib and lethal giant larvae homolog 2（Lgl2） was detected by Western blotting while the relative mRNA quantity of E-cadherin,N-cadherin,Occludin and junctional adhesion molecule-1（JAM1） was measured by realtime fluorescent quantitative polymerase chain reaction（RT-qPCR）.Results：XP induced a dose-dependent suppression on the proliferation of LoVo cells（P〈0.05 or P〈0.01）,with the inhibition rates varied from 27.30%to31.08%.Transwell assay showed that when preprocessed with PD98059 and XP-containing serum,the number of cells that passed the filter decreased significantly compared with that of NC group（P〈0.05 or P〈0.01）.Moreover,XP inhibited the protein expression of ERK1/2 and ZEB1（P〈0.05）;and up-regulated the protein expression of Scrib and Lgl2（P〈0.05）.The mRNA levels of E-cadherin,Occludin and JAM1 of the XP intervened groups and PC group markedly ascended（P〈0.05） while that of N-cadherin showed a descending tendency（P〉0.05）.Conclusion：XP intervention suppressed the ability of proliferation,invasion and migration of the LoVo cells.Regulating ZEB1-SCRIB Loop so as to recover epithelial phenotype and apical junctional complex might be one of the mechanisms by which XP produces the anti-metastasis effect.
基金Supported by the Beijing Traditional Chinese Medicine Science and Technology Project(QN2010-3)National Natural Science Foundation of China(No.81001564)
文摘OBJECTIVE： To investigate the anti-breast cancer （BC） effects and mechanisms of action of Xihuang pill （XHP） by conducting in vitro experiments on hu- man BC cell lines. METHODS： Two human BC cell lines （MCF-7 and MDA- MB231） were cultured and treated with XHP. Cell viability was detected using the 3-（4, 5-Dimeth- ylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay. Flow cytometry was used to measure the cell cycle and apoptosis. The cell cycle was ana- lyzed with propidium iodide staining. Apoptosis was evaluated using the Annexin V-fluorescein iso- thiocyanate/propidium iodide method. Western blotting was used to analyze the expression of es- trogen receptor （ER）-α and ER-13.RESULTS： XHP had growth-inhibitory effects on MCF-7 and MDA-MB231 cells with a half-maximal inhibitory concentration （IC50） of 10.14 mg/mL （MCF-7） and 8.98 mg/mL （MDA-MB231）. Apoptosis was induced to some extent. Certain changes in the ER were caused. Upregulation of ER-a protein was found in MCF-7 cells. ER-β expression in MDA-MB231 cells was increased. Cell-cycle arrest was not observed in the two BC cell lines. ER-β ex- pression in MCF-7 cells was unchanged. No ER-a ex- pression was shown in MDA-MB231 cells. CONCLUSION： These data suggest that XHP can af- fect cell viability and cause apoptosis, but that the cell cycle is not blocked. XHP has a certain impact on ER expression, but its mechanisms of action of anti-13C effects may not be due to regulation of ER expression.
文摘为了考察中药制剂西黄丸(Xihuang Pill)对肝癌细胞的作用及对Yes相关蛋白/具有PDZ结合基序的转录共激活因子(Yes-associated protein/transcriptional co-activator with PDZ-binding motif,YAP/TAZ)信号通路的影响,在SD大鼠中利用灌胃给药和动脉采血技术,制备西黄丸含药血清;利用磺酰罗丹明B(sulforhodamine B,SRB)染色技术,检测西黄丸含药血清对肝癌细胞增殖能力和克隆形成能力的影响;利用流式细胞术和原位末端转移酶标记(terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling,TUNEL)技术,检测西黄丸含药血清对肝癌细胞凋亡的影响;利用小室迁移测定实验模型,检测西黄丸含药血清对肝癌细胞侵袭和迁移能力的影响;利用小鼠肿瘤细胞异种移植模型,检测西黄丸对肝癌细胞体内增殖能力的影响;利用蛋白质印迹技术和荧光定量PCR技术,检测西黄丸含药血清对YAP/TAZ信号通路相关蛋白质和基因表达的影响。体外结果显示,西黄丸含药血清显著抑制肝癌细胞的体外增殖、克隆形成、侵袭和迁移能力,显著促进肝癌细胞的凋亡,显著抑制YAP/TAZ信号通路相关蛋白质和基因的表达,且具有剂量依赖性。体内结果显示,西黄丸显著抑制肝癌细胞的体内增殖能力,且具有剂量依赖性。研究结果提示,西黄丸能抑制肝癌细胞的体内外增殖、侵袭和转移能力,并可能在YAP/TAZ信号通路中发挥作用。
