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.

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.

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.

scholarly journals Botanical from Piper capense Fruit Can Help to Combat the Melanoma as Demonstrated by In Vitro and In Vivo Studies

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Brice E. N. Wamba ◽  
Paramita Ghosh ◽  
Armelle T. Mbaveng ◽  
Sayantan Bhattacharya ◽  
Mitra Debarpan ◽  
...  
Keyword(s):  
Western Blot ◽  
Melanoma Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Vasculogenic Mimicry ◽  
In Vivo Studies ◽  
Mesenchymal Transition

Piper capense belongs to Piperaceae family and has long been used as a traditional medicine to treat various diseases in several parts of Africa. The present study aims to investigate the effect of Piper capense fruit extract (PCFE) alone and in combination with dacarbazine on metastatic melanoma cell line B16-F10 and in vivo in C57BL/6J mice. Cytotoxic effects of PCFE alone and in association with dacarbazine on B16-F10 cells were studied by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and colony formation assay. Wound healing assay, immunofluorescence staining, and western blot analysis were performed to evaluate the individual and combined effect of PCFE and dacarbazine on epithelial-mesenchymal transition (EMT). For in vivo studies, C57BL/6J mice were subcutaneously injected with B16-F10 cells (5 × 105 cells/mL), and the effect of PCFE and dacarbazine was studied on tumor development. The alteration of EMT was evaluated by targeting E-cadherin, vimentin, and CD133 in PCFE alone and in combination with dacarbazine-treated tumor tissues by western blot analysis. Phytochemical screening of PCFE reveals the presence of certain secondary metabolites. Our results showed that PCFE alone and in association with dacarbazine has a good activity in preventing B16-F10 melanoma cell progression and clonogenicity. This extract also regulated EMT. In vivo results showed that PCFE (100 mg/kg body weight) reduced tumor size in C57BL/6J mice along with the decrease in the expression of vasculogenic mimicry (VM) tubes as well as an improvement in the qualitative and quantitative expression of markers involved in EMT. Our study suggests that PCFE may be useful for managing the growth and metastasis of melanoma.

scholarly journals Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Weisen Wang ◽  
Zhi Wang ◽  
Dingyuan Tian ◽  
Xi Zeng ◽  
Yangdong Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Notch Signaling ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Notch Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Integrin Β3 ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

TIPE1 suppresses the invasion and migration of breast cancer cells and inhibits epithelial-to-mesenchymal transition primarily via the ERK signaling pathway

2019 ◽  
Vol 51 (10) ◽  
pp. 1008-1015 ◽  
Author(s):  
Shusheng Qiu ◽  
Wei Hu ◽  
Qiuhong Ma ◽  
Yi Zhao ◽  
Liang Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Mesenchymal Transition ◽  
Invasion And Migration ◽  
And Migration

Abstract Tumor necrosis factor α-induced protein 8-like-1 (TIPE1) functions as an activator or a repressor in a tumor cell type-specific manner. However, the role of TIPE1 in breast cancer, especially regarding metastasis, is unknown. In this study, we aimed to investigate the TIPE1 expression in breast cancer tissues, the biological functions, and the underlying mechanisms of TIPE1 regarding the metastatic properties of breast cancer cells. The results of immunohistochemical staining and western blot analysis indicated that TIPE1 expression was associated with tumor size and lymph node metastasis, and the expression of TIPE1 was downregulated in the tissues of patients with lymph node metastasis. Transwell and wound healing assay results showed that TIPE1 inhibited the invasive and migratory capacities of breast cancer cells. Moreover, the epithelial-mesenchymal transition (EMT) was suppressed in TIPE1-overexpressing cells, as demonstrated by western blot analysis. In addition, western blot analysis also showed that TIPE1 reduced the expression levels of MMP2 and MMP9 and decreased the phosphorylation level of ERK. These results suggested that TIPE1 might suppress the invasion and migration of breast cancer cells and inhibit EMT primarily via the ERK signaling pathway. Our findings revealed the anti-tumor metastasis role of TIPE1 in breast cancer and TIPE1 might be a new candidate prognostic indicator and a potential molecular target for the treatment of breast cancer.

scholarly journals The Synergistic Anti-Cancer Effects of NVP-BEZ235 and Regorafenib in Hepatocellular Carcinoma

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2454
Author(s):  
Cheng-Chan Yu ◽  
Sung-Ying Huang ◽  
Shu-Fang Chang ◽  
Kuan-Fu Liao ◽  
Sheng-Chun Chiu
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Kinase Inhibitor ◽  
Epithelial Mesenchymal Transition ◽  
Combined Treatment ◽  
Gelatin Zymography ◽  
Migration And Invasion ◽  
Therapeutic Effects ◽  
Mesenchymal Transition ◽  
Anti Cancer

Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide. Regorafenib is a multi-kinase inhibitor and the second-line treatment for HCC. Since the PI3K/Akt/mTOR signaling pathway is dysregulated in HCC, we evaluated the therapeutic effects of regorafenib combined with a dual PI3K/mTOR inhibitor BEZ235 in the human HCC cell lines (n = 3). The combined treatment with BEZ235 and regorafenib enhanced the inhibition of cell proliferation and increased the expression of cleaved caspase-3 and cleaved PARP in HCC cells. Moreover, the combined treatment suppressed HCC cell migration and invasion in the transwell assay. Further, the Western blot analyses confirmed the involvement of epithelial-mesenchymal transition (EMT)-related genes such as slug, vimentin, and matrix metalloproteinase (MMP)-9/-2. Additionally, the proteinase activity of MMP-9/-2 was analyzed using gelatin zymography. Furthermore, the inhibition of phosphorylation of the Akt, mTOR, p70S6K, and 4EBP1 after combined treatment was validated using Western blot analysis. Therefore, these results suggest that the combined treatment with BEZ235 and regorafenib benefits patients with HCC.

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.

scholarly journals Identification of Tisp40 as an Essential Regulator of Renal Tubulointerstitial Fibrosis via TGF-β/Smads Pathway

2017 ◽  
Vol 42 (2) ◽  
pp. 697-712 ◽  
Author(s):  
Cheng-cheng Xiao ◽  
Jie Zhang ◽  
Peng-cheng Luo ◽  
Cong Qin ◽  
Yang Du ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Renal Fibrosis ◽  
Molecular Mechanisms ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Tubulointerstitial Fibrosis ◽  
Mesenchymal Transition ◽  
Renal Tubulointerstitial Fibrosis

Background: Tisp40, a transcription factor of the CREB/CREM family, is involved in cell proliferation, differentiation and other biological functions, but its role in renal tubulointerstitial fibrosis is unknown. Methods: In our study, we investigated the effects of Tisp40 on extracellular matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT) and the underlying molecular mechanisms in transforming growth factor-β (TGF-β)-stimulated TCMK-1 cells by quantitative real-time polymerase chain reaction (qPCR), Western blot analysis and immunofluorescence in vitro, and further explored the role of Tisp40 on renal fibrosis induced by ischemia-reperfusion (I/R) by qPCR, Western blot analysis, hydroxyproline analysis, Masson trichrome staining and immunohistochemistry staining in vivo. Results: The data showed that Tisp40 was upregulated in a model of renal fibrosis induced by I/R injury (IRI). Upon IRI, Tisp40-deficient mice showed attenuated renal fibrosis compared with wild-type mice. Furthermore, the expression of α-smooth muscle actin, E-cadherin, fibronectin, and collagen I was suppressed. Tisp40 overexpression aggravated ECM accumulation and EMT in the TGF-β-stimulated TCMK-1 cell line, whereas the opposite occurred in cells treated with small interfering RNA (siRNA) targeting Tisp40. Importantly, it is changes in the Smad pathway that attenuate renal fibrosis. Conclusion: These findings suggest that Tisp40 plays a critical role in the TGF-β/ Smads pathway involved in this process. Hence, Tisp40 could be a useful therapeutic target in the fight against renal tubulointerstitial fibrosis.

scholarly journals Ursolic Acid Inhibits Epithelial-Mesenchymal Transition through the Axl/NF-κB Pathway in Gastric Cancer Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jinxia Li ◽  
Chunyan Dai ◽  
Li Shen
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Western Blot ◽  
Cancer Cells ◽  
Ursolic Acid ◽  
Epithelial Mesenchymal Transition ◽  
Xenograft Model ◽  
Gastric Cancer Cells ◽  
Mesenchymal Transition

Background. Ursolic acid (UA) is an antitumor component derived from Chinese herbal medicine; this study is to observe the effects of UA on epithelial-mesenchymal transition (EMT) in gastric cancer. Methods. (1) In vitro experiments: 25μmol/L and 50μmol/L UA were applied to BGC-823, AGS, MGC-803, and HGC-27 cells; MTT staining, Transwell assay, and flow cytometry were used to assess cell proliferation, cell migration, and apoptosis, respectively. Western blot was performed to detect the expressions of N-Cadherin, Vimentin, Snail, Twist, Axl, p-Axl, IKK, p-IKK, NF-κB, and p-NF-κB. (2) In vivo experiments: Ten BALB/c-nu mice were used to establish gastric cancer xenograft model. Five were orally given UA for 4 weeks and five were given normal saline. Expressions of N-Cadherin and Snail were examined by immunohistochemical assay; expressions of N-Cadherin, Snail, Twist, Axl, p-Axl, IKK, and p-IKK were detected by Western blot. Results. (1) UA inhibited cell proliferation in BGC-823 and HGC-27 cells in dose-dependent manners. (2) UA inhibited cell migration in BGC-823, AGS, and MGC-803 cells while inducing apoptosis in BGC-823 cells. (3) UA significantly decreased the expressions of N-Cadherin, Vimentin, Snail, Twist p-Axl, p-IKKα/β, and p-NF-κB in BGC-823 and MGC-803 cells. (4) UA distinctly decreased the expressions of N-Cadherin, Snail, p-Axl, and p-IKKα/β in gastric cancer xenograft model rats. Conclusion. UA can effectively inhibit the proliferation and migration and induce apoptosis of gastric cancer cells. The antitumor effect of UA is conducted by EMT inhibition, which may be associated with the regulation of Axl/NF-κB signaling 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.

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