scholarly journals Verbascoside Inhibits Glioblastoma Cell Proliferation, Migration and Invasion While Promoting Apoptosis Through Upregulation of Protein Tyrosine Phosphatase SHP-1 and Inhibition of STAT3 Phosphorylation

2018 ◽  
Vol 47 (5) ◽  
pp. 1871-1882 ◽  
Author(s):  
Wei-Qiang Jia ◽  
Zhao-Tao Wang ◽  
Ming-Ming Zou ◽  
Jian-Hao Lin ◽  
Ye-Hai Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mouse Model ◽  
Western Blot ◽  
Protein Tyrosine Phosphatase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tyrosine Phosphatase ◽  
Migration And Invasion ◽  
Glioblastoma Cell ◽  
Stat3 Phosphorylation

Background/Aims: As a natural antioxidant, verbascoside (VB) is proved to be a promising method for the treatment of oxidative-stress-related neurodegenerative diseases. Thus, this study aimed to investigate the effects of VB on glioblastoma cell proliferation, apoptosis, migration, and invasion as well as the mechanism involving signal transducer and activator of transcription 3 (STAT3) and Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). Methods: U87 cells were assigned to different treatments. The MTT assay was used to test cell proliferation, flow cytometry was used to detect cell apoptosis, and a Transwell assay was used for cell migration and invasion. We analyzed the glioblastoma tumor growth in a xenograft mouse model. Western blot analysis was employed to determine the protein expression of related genes. Results: Glioblastoma cells exhibited decreased cell proliferation, migration, invasion, and increased apoptosis when treated with VB or TMZ. Western blot analysis revealed elevated SHP-1 expression and reduced phosphorylated (p)-STAT3 expression in glioblastoma cells treated with VB compared with controls. Correspondingly, in a xenograft mouse model treated with VB, glioblastoma tumor volume and growth were decreased. Glioblastoma xenograft tumors treated with VB showed elevated SHP-1, Bax, cleaved caspase-3, and cleaved PARP expression and reduced p-STAT3, Bcl-2, survivin, MMP-2, and MMP-9 expression. siRNA-SHP-1 inhibited the VB effects on glioblastoma. Conclusion: This study demonstrates that VB inhibits glioblastoma cell proliferation, migration, and invasion while promoting apoptosis via SHP-1 activation and inhibition of STAT3 phosphorylation.

Overcoming resistance to antiandrogens with a TGF-β RI inhibitor in preclinical mouse model of PCa.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 288-288
Author(s):  
Channing Judith Paller ◽  
Hong Pu ◽  
Diane Begemann ◽  
Mary Nakazawa ◽  
Natasha Kyprianou
Keyword(s):  
Cell Proliferation ◽  
Mouse Model ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Combined Treatment ◽  
Prostate Tumor ◽  
Metastatic Progression ◽  
Mesenchymal Transition

288 Background: Epithelial-mesenchymal transition (EMT) is a significant contributor to PCa metastatic progression and therapeutic resistance in patients treated with the androgen receptor (AR) directed therapies. We previously demonstrated that aberrant TGF-β signaling accelerates prostate tumor progression in the TRAMP mouse model of tumorigenesis via selective effects on EMT. Methods: We hypothesize that the combination of the TGF-β receptor inhibitor, galunisertib (G), and enzalutamide (E) will perturb the interactive signaling between TGF-β and AR signaling affecting the phenotypic landscape of EMT. This perturbation may be exploited in our mouse model, towards enhanced anti-tumor efficacy in advanced castration-resistant PCa (CRPC). We treated 2-week old mice for two weeks with the G (75mg/kg) and/or E (30mg/kg) in combination and as single agents. Results: Treatment with G alone or in combination with E resulted in a significant reduction in prostate tumor weight without affecting total body weight. Immunohistochemical (IHC) and Western blot analysis showed that, while treatment with the G alone led to increased apoptosis and decreased cell proliferation, combination of G and E had significantly higher efficacy in inducing apoptosis and inhibiting cell proliferation than either E or G alone. As expected treatment with the G decreased the levels of nuclear Smad4 protein; the combination of G and E further decreased nuclear Smad4 expression. Furthermore the combination of G and E reversed phenotypic EMT to MET (mesenchymal-epithelial-transition), as assessed by the increase in E-cadherin among the prostate tumor cell populations. IHC and Western blot analysis also revealed that the combined treatment of G and E led to a significant decrease in nuclear AR levels compared to E-only-treated or vehicle-control tumors. Conclusions: These results provide significant insights as to the therapeutic impact of G to effectively impair the TGF-β signaling and overcome resistance of PCa patients to E by reversing EMT to potentially sensitize tumors to the antiandrogen effect. This study has major translational relevance; the combination of G and E may lead to synergistic anti-tumor impact in patients with CRPC.

scholarly journals Demethylation of TIMP2 and TIMP3 inhibit cell proliferation, migration and invasion in pituitary adenoma

2019 ◽  
Author(s):  
Yongdong Yang ◽  
Fanjun Huang ◽  
Xiufu Wu ◽  
Chunqin Huang ◽  
Shenyu Li
Keyword(s):  
Dna Methylation ◽  
Cell Proliferation ◽  
Pituitary Adenoma ◽  
Western Blot ◽  
Promoter Methylation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Gh3 Cells ◽  
Migration And Invasion

Abstract Background: Pituitary adenoma (PA) is one of the most common intracranial neoplasms. Tissue inhibitors of metalloproteinases (TIMPs) are prognostic biological markers, but their biological roles remains largely unclear in invasive PA. Methods: The promoter methylation status of TIMP2 and TIMP3 genes in invasive PA tissues and cells was measured by methylation-specific polymerase chain reaction (MSP). The expression of TIMP1-3 was validated by quantitative real time PCR and western blot analysis. Overexpression and knockdown of TIMP2 and TIMP3 in GH3 cells were created by transfection of pcDNA3.0 and siRNA against TIMP2 and TIMP3, respectively. Functional experiments in GH3 cells were performed with CCK-8 assay, wound healing assay and transwell assay. Effects of 5-Azacytidene (5-AzaC) on the methylation of TIMP2 and TIMP3 gene, and DNA methyltransferase 1 (DNMT1), DNMT3a and DNMT3b were determined by western blot analysis. Results: We found the expression of TIMP1, TIMP2 and TIMP3 was down-regulated in invasive PA tissues and cells. Moreover, decreased TIMP2 and TIMP3 expression was closely associated with their promoter methylation. The in vitro experiments showed that overexpression of TIMP2 and TIMP3 exerted suppressive effects, while knockdown of TIMP2 and TIMP3 presented enhanced effects on cell proliferation, migration and invasion in PA cells. Furthermore, 5-AzaC treatment concomitantly up-regulated the protein levels of TIMP2, TIMP3, DNMT1, DNMT3a and DNMT3b. Conclusions: In conclusion, our results support that DNA methylation at least partly accounts for TIMP2 or TIMP3 silencing in invasive PA, which will provide new insights into the mechanisms underlying the function of TIMPs in PA. Key words: Invasive pituitary adenoma, TIMPs, DNA methylation, 5-AzaC, GH3

scholarly journals Demethylation of TIMP2 and TIMP3 inhibit cell proliferation, migration and invasion in pituitary adenoma

2019 ◽  
Author(s):  
Yongdong Yang ◽  
Fanjun Huang ◽  
Xiufu Wu ◽  
Chunqin Huang ◽  
Shenyu Li
Keyword(s):  
Cell Proliferation ◽  
Pituitary Adenoma ◽  
Western Blot ◽  
Promoter Methylation ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Methylation Status ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Gh3 Cells ◽  
Migration And Invasion

Abstract Background: Pituitary adenoma (PA) is one of the most common intracranial neoplasms. Tissue inhibitors of metalloproteinases (TIMPs) are prognostic biological markers, but their biological roles remains largely unclear in invasive PA. Methods: The promoter methylation status of TIMP2 and TIMP3 genes in invasive PA tissues and cells was measured by methylation-specific polymerase chain reaction (MSP). The expression of TIMP1-3 was validated by quantitative real time PCR and western blot analysis. Overexpression and knockdown of TIMP2 and TIMP3 in GH3 cells were created by transfection of pcDNA3.0 and siRNA against TIMP2 and TIMP3, respectively. Functional experiments in GH3 cells were performed with CCK-8 assay, wound healing assay and transwell assay. Effects of 5-Azacytidene (5-AzaC) on the methylation of TIMP2 and TIMP3 gene, and DNA methyltransferase 1 (DNMT1), DNMT3a and DNMT3b were determined by western blot analysis. Results: We found the expression of TIMP1, TIMP2 and TIMP3 was down-regulated in invasive PA tissues and cells. Moreover, decreased TIMP2 and TIMP3 expression was closely associated with their promoter methylation. The in vitro experiments showed that overexpression of TIMP2 and TIMP3 exerted suppressive effects, while knockdown of TIMP2 and TIMP3 presented enhanced effects on cell proliferation, migration and invasion in PA cells. Furthermore, 5-AzaC treatment concomitantly up-regulated the protein levels of TIMP2, TIMP3, DNMT1, DNMT3a and DNMT3b. Conclusions: In conclusion, our results support that DNA methylation at least partly accounts for TIMP2 or TIMP3 silencing in invasive PA, which will provide new insights into the mechanisms underlying the function of TIMPs in PA.

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

Lidocaine Inhibits Ovarian Cancer Cell Proliferation and Induces Apoptosis: An In Vitro Study

2020 ◽  
Vol 10 (5) ◽  
pp. 724-729
Author(s):  
Yaping Xu ◽  
Xiaoqin Fang ◽  
Xianjiang Wei
Keyword(s):  
Ovarian Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Group Treatment ◽  
Western Blot Analysis ◽  
Ovarian Cancer Cell ◽  
Control Group

Objective: The present study aimed to explore the effects and related mechanism of lidocaine on human ovarian cancer cell lines. Methods: Human ovarian cancer cell lines (SKOV3 and ES-2) were treated with different concentrations of lidocaine for different time. We treated SKOV3 and ES-2 cells using lidocaine then used MTT assay and flow cytometry to detect the cell proliferation and cell apoptosis. In addition, we used western blot analysis to explore the protein expression of Bax and Bcl-2 in SKOV3 and ES-2 cells. Western blot analysis and qRT-PCR were performed for the detection of EMT markers (E-cadherin, N-cadherin). The protein expression levels of TRAF3 and p-p65 in SKOV3 and ES-2 cells were determined by Western blot analysis. Results: Compared to the control group, 0.5, 1, 5, and 10 mM of lidocaine significantly inhibited ovarian cancer cell proliferation at different time points, while 0.1 mM of lidocaine had no significant effect. 1, 5 mM of lidocaine induced the cell apoptosis, and observably reduced expression of Bcl-2 protein, but improved Bax expression markedly compared with the control group. Treatment of lidocaine increased E-cadherin expression, but decreased N-cadherin expression when compared with control group. Treatment of lidocaine increased TRAF3 protein expression, but decreased p-p65 protein expression in ES-2 and SKOV3 cells. Conclusion: We demonstrated that lidocaine inhibited cell proliferation, induced apoptosis, and inhibited EMT in ovarian cancer cells via regulating TRAF3/NF-κB pathway.

miR-106b-5p, miR-17-5p to predict recurrence and progression in breast DCIS model based on TGFβ paradox.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23019-e23019
Author(s):  
Jieun Lee ◽  
Kwangil Yim ◽  
Dong-Min Kim ◽  
Young-Seok Song ◽  
Ahwon Lee
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ductal Carcinoma ◽  
Local Treatment ◽  
Proliferation Index ◽  
Transfected Cell Line ◽  
Mcf 7

e23019 Background: Ductal carcinoma in situ (DCIS) is a well-known precursor of invasive ductal carcinoma (IDC). Part of patients show disease recurrence as DCIS or IDC after local treatment, but there are no established markers for prediction of recurrence. Methods: Authors analyzed 30 patients diagnosed as pure DCIS, recurrent DCIS, and IDC progressed in DCIS background. miRNA was extracted from archival tissue, and hierarchical clustering of miRNA microarray was performed. We selected highly expressed miR-17-5p and miR-106b-5p as marker for recurrence of DCIS. Two miRNAs were transfected to MCF-12 and MCF-7 cell line. Cell proliferation assay and Western blot analysis was performed for analyzing the interaction between cell proliferation and TGFβ downstream pathway. Results: miR-106b-5p single and combined miR-106b-5p and miR-17-5p transfected MCF-12 cell line showed increased proliferation index compared to un-transfected cell line. In MCF-7, miR-106b and miR-17-5p transfected cell line showed inferior proliferation index compared to un-transfected cell line. Western blot analysis showed minimal increased expression of SMAD4, phosphorylated SMAD2 (pSMAD2) in miR-106b-5p and miR-17-5p transfected MCF-12 cell line. However, decreased expression of TGFBR2 and no interval change of SMAD4 and pSMAD2 was detected in miR-106b-5p and miR-17-5p transfected MCF-7 cell line. Conclusions: miR-106-5p, miR-17-5p showed increased expression in recurrent DCIS or IDC based on miRNA hierarchical microarray. miRNA transfected MCF-12 cell line showed increased proliferation index and activated TGFβ downstream pathway. miRNA transfection might have made normal cell line to pre-cancerous cell line and TGFβ pathway might have influenced to promote tumor proliferation, based on TGFβ paradox hypothesis.

scholarly journals Downregulation of Tim-1 inhibits the proliferation, migration and invasion of glioblastoma cells via the miR-133a/TGFBR1 axis and the restriction of Wnt/β-catenin pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Li Wei ◽  
Ya Peng ◽  
Naiyuan Shao ◽  
Peng Zhou
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Binding Sites ◽  
Migration And Invasion ◽  
Normal Brain ◽  
Glioblastoma Cell ◽  
Rna Seq ◽  
Invasion And Migration ◽  
And Migration ◽  
U87 Cells

Abstract Background Glioblastoma remains one of the most lethal brain cancers. T-cell immunoglobulin and mucin domain 1 (Tim-1) is associated with various immune diseases. The molecular mechanism of Tim-1 in regulating glioblastoma cell proliferation, invasion, and migration is still unknown. Moreover, it has shown that miR-133a plays an important role in glioblastoma. However, little is known about the interaction between Tim-1 and miR-133a in glioblastoma. Methods Tim-1 expression in glioblastoma and normal brain tissues was detected by qPCR, Western Blot and IHC. After Tim-1 knockdown in U251 and U87 cells, genes showing significantly differential expression, along with the significant differential miRNAs were analyzed using RNA-seq analysis. The binding sites were verified using dual-luciferase reporter gene assay. U251 and U87 cells were allocated into the small harpin-negative control (sh-NC), sh-Tim-1, sh-Tim-1 + inhibitor NC, and sh-Tim-1 + miR-133a inhibitor group. Cell proliferation, migration, and invasion were determined by CCK-8, flow cytometry, wound-healing and Transwell assays, respectively. Next, U251 and U87 cells were allocated into the mimic NC, miR-133a mimic, miR-133a mimic + pcDNA3.1, and miR-133a mimic + pcDNA3.1-TGFBR1 groups, followed by the detection of cell proliferation, migration, and invasion. Western blot was used to identify the expression of vital kinases in the Wnt/β-catenin pathway. Results Tim-1 was highly expressed in glioblastoma tissues compared with that in normal brain tissues. RNA-seq analysis showed that Tim-1 knockdown could lead to the downregulation of TGFBR1 and the upregulation of miR-133a. The binding sites between TGFBR1 and miR-133a were confirmed. Tim-1 knockdown impaired the invasion, migration, proliferation of U251 and U87 cells, which could be reversed by miR-133a downregulation. miR-133a upregulation inhibited the proliferation, invasion, and migration of U251 and U87 cells, which could be reversed by TGFBR1 upregulation. Tim-1 knockdown and miR-133a upregulation could inhibit the activation of the Wnt/β-catenin pathway, while the elevation of TGFBR1 showed opposite effects. Conclusion Tim-1 knockdown inhibited glioblastoma cell proliferation, invasion, and migration through the miR-133a/TGFBR1 axis and restrained the activation of the Wnt/β-catenin pathway.

scholarly journals ARHGEF12 Is Essential for Human Megakaryocyte Differentiation and Plays Critical Roles in Platelet Function

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 341-341
Author(s):  
Siying Zou ◽  
Alexandra M Teixeira ◽  
Chad D Sanada ◽  
Ping-xia Zhang ◽  
Diane Krause
Keyword(s):  
Platelet Aggregation ◽  
Mouse Model ◽  
Western Blot ◽  
Platelet Activation ◽  
Platelet Function ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Thrombin Receptors ◽  
Platelet Functions

Abstract Megakaryocytopoiesis, the process by which hematopoietic stem cells develop into mature megakaryocytes (MK), and thrombopoiesis, platelet production/release, are critical for blood homeostasis. We tested the hypothesis that the Rho guanine exchange factor, ARHGEF12 (also known as LARG), is critical for MK differentiation and platelet functions based on the following: 1) ARHGEF12 is part of a recurrent translocation with MLL in acute myeloid leukemia. 2) Both published microarray datasets and deep-sequencing data from our lab on primary human CD34+ cells differentiating into MKs show that ARHGEF12 expression goes up dramatically during MK differentiation. 3) ARHGEF12 is one of the most highly expressed guanine exchange factors in platelets. 4) ARHGEF12 forms a complex with G proteins and stimulates Rho-dependent signals. It is known that platelet activation can be initiated by extracellular stimuli working through G protein-coupled receptors and Rho signaling, suggesting that ARHGEF12 may function in platelet activation. 5) Mice with KO of RhoA (a known ARHGEF12 substrate) in the MK-lineage have macrothrombocytopenia and defective platelet activation. To test this hypothesis, we used ARHGEF12 shRNA mediated KD and an ARHGEF12 specific pharmacological inhibitor (Y16) in both murine and human primary cells, and characterized a LARG KO mouse model for MK and platelet phenotypes, and found: ARHGEF12 is differentially upregulated during MK differentiation and is enriched in platelets Using quantitative RT-PCR and western blot analysis at different timepoints of primary FACSorted Mk progenitors induced to differentiate into mature MK in vitro, ARHGEF12 RNA and protein expression increases during MK differentiation in both the murine and human systems. Also western blot analysis of murine platelet rich plasma shows that ARHGEF12 protein is highly expressed in platelets. ARHGEF12 is essential for human MK differentiation To test the function of ARHGEF12 in Mk differentiation, we used lentiviral shRNA to knockdown ARHGEF12 in FACSorted primary human Mk progenitors from mobilized peripheral blood differentiated in vitro to MK. The results show that ARHGEF12 knockdown blocks MK polyploidization (not shown) and maturation (Fig. A). This was confirmed using a published ARHGEF12 inhibitor (Y16) in the differentiation culture of human MK progenitors, in which there was a dose-dependent block in MK differentiation (Fig. B). These data suggested that ARHGEF12 is essential for human MK differentiation. We researched the function of ARHGEF12 in the murine system using a constitutive ARHGEF12 knockout mouse model. The mice have enlarged platelets (p=0.07) and a decreased platelet count (p=0.01). However, the knockout mice have normal BM cellularity with no change in megakaryocyte number or ploidy, suggesting that ARHGEF12 is dispensable for murine MK differentiation in vivo. ARHGEF12 is essential for platelet function in both the murine and human systems: To test whether ARGEF12 functions in platelet activation, we compared WT versus KO platelet activation in vitro. We tested activation in response to ADP, U46619 (Thromboxane), ADP+U46619, and Thrombin. KO plateelts have significantly reduced activation in response to U46619 and thrombin, with no effects on ADP-induced activation. Analogous studies using the ARHGEF12 inhibitor (Y16) on WT platelets revealed supportive evidence. Lastly, we tested ARHGEF12 function in human platelet aggregation using the Y16 compound. Consistent with the murine data, Y16 blocked platelet aggregation in response to both U46619 and Thrombin. Taken together, these data strongly suggest that ARHGEF12 is essential for platelet function and acts downstream of the Thromboxane and Thrombin receptors. In summary, we found that ARHGEF12 is differentially up-regulated in MK differentiation both in human and in mouse system,. It plays a critical role in human Mk differentiation but is dispensable in murine MK differentiation, and ARHGEF12 is critical for platelet functions in both human and mouse systems, potentially acting downstream of Thromboxane and Thrombin receptors. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract 1252: A Mouse Model of a Familial Dilated Cardiomyopathy Mutation in Titin Recapitulates the Human Phenotype

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Michael Gramlich ◽  
Beate Michely ◽  
Brenda Gerull ◽  
Christian Krohne ◽  
Ingo Morano ◽  
...  
Keyword(s):  
Mouse Model ◽  
Western Blot ◽  
Dilated Cardiomyopathy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Autosomal Dominant ◽  
Premature Stop Codon ◽  
Left Ventricular ◽  
Insertion Mutation ◽  
Human Phenotype

Dilated cardiomyopathy (DCM) is the most common form of primary myocardial diseases and the third most common cause of heart failure. Familial occurrence, mostly as an autosomal dominant trait, is responsible for 20 –30% of all DCM cases. We have previously shown that mutations in the giant muscle filament titin ( TTN ) cause dilated cardiomyopathy. In a large DCM kindred (A1) with autosomal dominant inherited DCM, we could identify a 2 bp insertion mutation in exon 326 of TTN . This heterozygous nonsense mutation leads to a framshift generating a premature stop codon after the addition of 4 novel amino acid residues. We have recently evaluated a cardiac biopsy sample from an affected family member of kindred A1 showing that no truncated protein is observed in a western blot analysis. To further investigate the functional consequences of the identified human TTN mutation, we now generated a mouse model that includes the 2bp insertion at the corresponding site in the mouse genome. Heterozygous mice are viable and fertile. As in the human situation, the truncated titin is not detectable in western blot analysis of cardiac tissue indicating haploinsufficiency. The ventricles of the heterozygous animals show a decrease in ventricular stiffness as seen in isolated working heart pressure measurements and transmitral Doppler echocardiography (E:A 1.34 vs. 1.075, p<0.01; IVRT 13.57ms vs. 17.01ms, p<0.05). When exposed to angiotensin II (1.4 mg/kg/d for 14d) as a cardiac stressor, heterozygous animals develop dilatation of the left ventricles (4.45mm vs. 3.77 mm, p<0.05) with impaired fractional shortening (25.12% vs. 32.86%, p<0.01) and a diffuse myocardial fibrosis. Homozygous mice die in utero before E8.0. Whether a defect in the formation of sarcomeres or, alternatively, a defect in yet unknown non-muscle functions of titin account for this early embryonic lethality remains to be determined. Conclusion: Our mouse model shows that a mutation in TTN leads to impaired biomechanical properties of the heart, resulting in left ventricular dilatation and decreased systolic function, thereby recapitulating the human phenotype.

HER4 promotes the growth and metastasis of osteosarcoma via the PI3K/AKT pathway

2020 ◽  
Vol 52 (4) ◽  
pp. 345-362 ◽  
Author(s):  
Xiaodong Li ◽  
Qingshan Huang ◽  
Shenglin Wang ◽  
Zhen Huang ◽  
Fengqiang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Osteosarcoma Cells ◽  
Her4 Protein

Abstract Osteosarcoma is the most common primary malignant bone tumor, which occurs in adolescents. As reported by our previous studies, HER4 indicates a poor prognosis of primary osteosarcoma. However, its mechanisms in the pathogenesis of osteosarcoma have not yet been studied. The purpose of this study was to investigate the role of HER4 in osteosarcoma and whether the PI3K/AKT pathway is involved. In this study, western blot analysis was used to investigate the expression of HER4 protein in osteosarcoma tissues and cell lines. CCK8 and transwell assays were used to detect the effects of HER4 on the proliferation, migration, and invasion of osteosarcoma cells in vitro. The effects of HER4 on the growth and metastasis of osteosarcoma in vivo were detected by tumor formation and immunofluorescence in nude mice. The role of the PI3K/AKT pathway in HER4 regulation of the growth and metastasis of osteosarcoma was examined by western blot analysis and immunofluorescence assay. We found that HER4 protein was highly expressed in clinical osteosarcoma specimens and osteosarcoma cells. HER4 markedly promoted the proliferation, migration, and invasion of osteosarcoma cells in vitro as well as the growth and metastasis of osteosarcoma in vivo. HER4 overexpression upregulated the expression of phosphorylated protein kinase B (pAKT), proliferation marker antigen Ki67, and metastasis cell marker matrix metalloproteinase 9 (MMP9). Notably, PI3K/AKT inhibitor LY294002 significantly inhibited the effects of HER4 via the downregulation of pAKT, Ki67, and MMP9. Moreover, LY294002 markedly blocked the effects of HER4-induced upregulation of tumor malignancy. The present study suggests that HER4 may promote the growth and metastasis of osteosarcoma via the PI3K/AKT pathway. The HER4/PI3K/AKT pathway could serve as a potential target for the treatment of osteosarcoma.

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