C-jun N-terminal kinase dependent autophagic cell death in cancer cells induced by Zanthoxylum fruit extract from Japanese pepper.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 653-653
Author(s):  
Toru Kono ◽  
Reo Nozaki ◽  
Hiroki Bochimoto ◽  
Tsuyoshi Watanabe ◽  
Kaori Oketani ◽  
...  
Keyword(s):  
Cell Death ◽  
Drug Development ◽  
Western Blot ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Fruit Extract ◽  
Cytoplasmic Vacuolization

653 Background: Natural products constitute a promising resource for drug development including an anticancer drug. Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) is an important component of Daikenchuto, which is a form of Japanese traditional medicine. Recently, we have reported that Daikenchuto has an anticancer activity in vivo, however precise mechanism is still unclear. Therefore, we investigated the potential anticancer activity of ZFE as an inducer of autophagic cell death (ACD). Methods: ZFE powder was provided by Tsumura (Japan). We investigated the effect of ZFE on the morphology of six types of human cancer cells and normal cells by using phase contrast microscopy and electron microscopy. Knockdown of autophagy-related gene 5 (ATG5), which is an essential gene for autophagy, by transfecting small interfering RNA was performed and confirmed by quantitative RT-qPCR and Western blot analysis. Effect of bafilomycin A1 (Baf A1), an inhibitor of vacuolar type H+-ATPases, on the anticancer activity of ZFE was investigated. Western blot analysis revealed LC3-II levels, a marker of autophagy. Results: ZFE caused remarkable autophagy-like cytoplasmic vacuolization with the inhibition of cell proliferation and subsequent induction of cell death in human cancer cell lines, DLD-1, HepG2 and Caco-2 cells but not in A549, MCF-7 or WiDr cells. ZFE increased LC3-II protein levels. Suppression of an ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, ZFE increased the phosphorylation of c-jun N-terminal kinase (JNK) in cancer cells which can be induced cell death by ZFE and JNK inhibitor SP600125 attenuated both vacuolization and cell death induced by ZFE. Instead, ZFE-induced cell death was neither apoptosis nor necrosis according to the morphological perspective and the marker of apoptosis or necrosis. And normal intestinal cell was not affected by ZFE. Conclusions: ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Alessandro Bressan ◽  
Francesca Bozzo ◽  
Carlo Alberto Maggi ◽  
Monica Binaschi
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Tandem Repeat ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Ovarian Cancer Cells ◽  
Repeat Region ◽  
Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

scholarly journals Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells

2013 ◽  
Vol 33 (3) ◽  
Author(s):  
Mohammad K. Ghalayini ◽  
Qihan Dong ◽  
Des R. Richardson ◽  
Stephen J. Assinder
Keyword(s):  
Prostate Cancer ◽  
Epithelial Cells ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Metastasis Suppressor ◽  
Normal Prostate ◽  
Truncated Protein ◽  
Prostate Epithelial Cells

NDRG1 (N-myc downstream regulated gene-1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and Western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag (phosphate-binding tag) SDS/PAGE assay, demonstrated that the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed that the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT–PCR (reverse transcriptase–PCR) demonstrated only a single amplicon, and thus, the two bands could not result from an alternatively spliced NDRG1 transcript. Western-blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with MS confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human PrECs (prostate epithelial cells). Western-blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting that it lacked N-terminus amino acids (residues 1–49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49–Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity.

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 Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Nutlin-3A, an MDM2 Antagonist, Induces p53-Dependent Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma (HL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2506-2506
Author(s):  
Elias Drakos ◽  
Athanasios Thomaides ◽  
Jiang Li ◽  
Marina Konopleva ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Small Molecule ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
P53 Gene ◽  
Cycle Arrest

Abstract p53 is the most frequently mutated tumor suppressor gene in human cancer. However, in Hodgkin lymphoma (HL) p53 is mutated only in a small subset of cases suggesting that modulation of wild-type-p53 (wt-p53) levels in Hodgkin and Reed-Sternberg (HRS) cells may have therapeutic implications in these patients. MDM2 (HDM2 in humans) is a physiologic negative regulator of p53 levels through a well-established auto-regulatory feedback loop. Nutlin-3A is a recently developed small molecule, which antagonizes mdm2 through disruption of p53-MDM2 interaction resulting in p53 stabilization. We hypothesized that nutlin 3A may stabilize p53 in HRS cells carrying wt-p53 gene, thus leading to p53-dependent apoptosis and G1-S cell cycle arrest. We used two novel classical HL cell lines recently established in our Institution, MDA-V and MDA-E, which have been shown to carry wt-p53 gene. As a control, we used a HL cell line L-428 harboring a mutant p53 (mt-p53) gene product (deletion at exon 4). We investigated effects on apoptosis and cell cycle arrest after treatment of cultured HRS cells with nutlin-3A or a 150-fold less active enantiomere, nutlin-3B. Treatment with nutlin-3A resulted in substantial cell death (up to 65%) in a concentration-dependent manner associated with increased apoptosis as shown by apoptotic morphology (DAPI immunofluorescence), annexin V binding (flow cytometry) and caspase activation (Western blot analysis) in MDA-V and MDA-E cells, but not in L-428 cells. Nutlin-3A-induced apoptotic cell death was accompanied by stabilization of p53 protein as detected by western blot analysis and immunofluorescence and up-regulation of pro-apoptotic Bax, a known target of p53. Inhibition of nuclear export by leptomycin B stabilized p53 at a similar level as compared to nutlin-3A treatment in these cells, suggesting that nutlin-3A stabilized p53 through inhibition of MDM2-mediated degradation of the protein. By contrast, no changes in cell viability, growth or apoptosis were seen after treatment with the inactive nutlin-3B small molecule. Treatment with nutlin-3A also resulted in a significant decrease (up to 85%) of cells in S-phase and a dose-dependent increase of cells in G1 phase of cell cycle as detected by flow cytometry, in MDA-V and MDA-E cells, but not in L-428 cells. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21, a transcriptional target of p53. In contrast, treatment of HRS cells with nutlin-3B had no effects on the cell cycle irrespective of p53 mutation status. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed synergistic effects and enhanced cytotoxicity in HRS cells with wt-p53 gene. Targeting MDM2 with the specific antagonist nutlin-3A that leads to non-genotoxic p53 activation, apoptosis induction and cell cycle inhibition may provide a new therapeutic approach for patients with HL.

HDAC and LSD1 Inhibitors Synergize to Induce Cell Death in Acute Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1427-1427 ◽  
Author(s):  
Lorimar Ramirez ◽  
Melissa Singh ◽  
Joya Chandra
Keyword(s):  
Cell Death ◽  
Monoamine Oxidase ◽  
Acute Leukemia ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines

Abstract Abstract 1427 Histone deacetylase inhibitors (HDACi) are a class of emerging epigenetic therapies which are being used to treat cancer. Two HDACi (vorinostat and romidepsin) are FDA approved for cutaneous T-cell lymphoma. HDACi have been employed in clinical trials for acute leukemia, but single agent activity has been limited. Improved efficacy is observed when combined with other anticancer agents. In the current study we addressed acute leukemia models using vorinostat, a pan-HDACi that inhibits HDAC class I, II, and IV and entinostat, a newer HDACi that inhibits HDAC class I more specifically. These HDACi were combined with inhibition of another histone modifying enzyme: lysine specific demethylase 1 (LSD1). The LSD1 gene encodes a favin-dependent monoamine oxidase, which demethylates mono- and di-methylated lysines, specifically lysines 4 and 9 on histone 3 (H3K4 and H3K9), thus it is also involved in gene regulation through post-translational histone modification. LSD1 overexpression has been linked to human carcinogenesis in bladder carcinomas, lung cancer, and poorly differentiated neuroblastoma. However, it has not been studied in hematologic malignancies. Because LSD1 is structurally similar to monoamine oxidase (MAO), it has been shown that nonselective MAO inhibitors also inhibit LSD1. Here we employed tranylcypromine, a monoamine oxidase inhibitor (MAOi), as an irreversible LSD1 inhibitor. Recently published work from our laboratory has shown synergistic effects of combined HDAC and LSD1 inhibition in brain tumors (glioblastoma multiforme). Similar results have been published in breast cancer cells, but no work has been done in hematological malignancies. The objective of this study was to investigate the possible synergy of HDAC and LSD1 inhibitors in acute leukemia cells. LSD1 protein expression in several leukemia cells lines was analyzed by Western blot analysis. LSD1 was expressed in all leukemia cell lines tested, which included T-cell ALL (Jurkat, Sub-T1, MOLT4), B-cell ALL (JM-1,697), and Philadelphia chromosome positive ALL (Z33, Z119, Z181). To determine whether synergy exists between HDACi and LSD1 inhibitors, Jurkat cells were exposed to different concentrations of tranylcypromine and vorinostat or entinostat. After 24 hr, DNA fragmentation was assessed by propidium iodide (PI) staining followed by flow cytometric analysis. A combination index (CI) less than 1.0 is representative of synergism as measured by Calcusyn software. Results showed a synergistic effect on DNA fragmentation when combining the 2.5 μM dose of vorinostat with a range of tranylcypromine doses (1 mM CI= 0.78, 1.5 mM CI= 0.49, and 2 mM CI= 0.39). The same effect was observed with the combination of 2.5 μM entinostat with 2 mM tranylcypromine (CI=0.52). Viability studies performed with the same drug concentrations in conbination also showed statistically significant cell death. Additional acute leukemia cell lines, 697 and MOLT-4, also demonstrated significantly increased cell death with the combination relative to treatment with either agent alone. Since these agents inhibit histone deacetylation and lysine demethylation, we tested whether these histone modifications were promoted by combination treatment. Jurkat cell lysates were generated by acid extraction of histones and Western blot analysis was conducted. We demonstrated that in fact histone acetylation was increased with combination treatment, indicating that these modifications coordinately regulate each other in acute leukemia cells. A molecular target for LSD1 is p53, a tumor suppressor protein whose activity is regulated by lysine methylation and demethylation. Western blot analysis showed that p53 is downregulated in leukemia cells after exposure to the combination of HDAC and LSD1 inhibitors. Future studies will address if p53 downregulation is a trigger for the synergistic cell death. Taken together, our data shows the efficacy of combining LSD1 inhibitors with HDAC inhibitors in multiple acute leukemia models. Since tranylcypromine is also a FDA-approved agent, these results urge the design of a feasible and effective clinical trial combining LSD1 and HDAC inhibitors for acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Androgen receptor and HIF-1α interaction in prostate cancer.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 197-197 ◽  
Author(s):  
Kelie M Reece ◽  
Sarah M Troutman ◽  
Heather M Pressler ◽  
Stephen T Pisle ◽  
William Douglas Figg
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
The United States ◽  
Tumor Resistance ◽  
Prostate Cancer Cells ◽  
Experimental Conditions

197 Background: Prostate cancer is the most common noncutaneous cancer among men in the United States, and its progression is largely controlled by the androgen receptor (AR). Androgen deprivation therapy (ADT) is an initially effective treatment for prostate cancer, but most tumors eventually become castrate resistant. Tumor hypoxia also appears to be associated with a poor prognosis in prostate cancer. HIF-1a regulates the transcription of genes that allow tumor survival and growth in low oxygen conditions. Our laboratory has data showing that in response to castration and anti-androgen therapy in mice, there was a strong transcriptional relationship between HIF-1a and AR, as measured by quantitative RT-PCR, suggesting an interaction between the two proteins. Thus, the purpose of this study was to determine if there is a molecular interaction between HIF-1a and AR in prostate cancer cells. Methods: We used Western blot analysis to examine the expression levels of HIF-1a and AR in LNCaP prostate cancer cells to determine if they are upregulated together at the protein level. Four experimental conditions were tested: control (no treatment), DHT for induction of AR expression, CoCl2 for induction of HIF-1a, and a combination treatment of DHT and CoCl2. Immunoprecipitation experiments were carried out to determine if there is an association between HIF-1a and AR. In addition, cells were fractionated into nuclear and cellular cells extracts, followed by Western blot analysis to determine where in the cells this interaction occurs. Results: Western blot analysis of cell lysates showed synergistic upregulation of both HIF-1a and AR expression only under combined CoCl2 and DHT treatment conditions. In addition, immunoprecipitation experiments showed that HIF-1a and AR exist in a complex with one another, and fractionation experiments indicated this complex occurs in the nucleus. Conclusions: Our results demonstrate that HIF-1a and AR associate with one another in cells. Binding assays are in progress to determine the nature of this interaction. In addition, we are examining the cellular consequences of this protein interaction, as it is possible that upregulation of HIF-1a in response to low androgen contributes to the development of tumor resistance to ADT.

scholarly journals Suppression of CCT3 inhibits the proliferation and migration in breast cancer cells

2020 ◽  
Author(s):  
Gang Xu ◽  
Shanshan Bu ◽  
Xiushen Wang ◽  
He Zhang ◽  
Hong Ge
Keyword(s):  
Breast Cancer ◽  
Signal Transduction ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Signal Transduction Proteins ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background CCT3 is a subunit of chaperonin-containing TCP-1 (CCT), which folds many proteins involved in cancer development and plays an important role in many cancers. However, the role of CCT3 in breast cancer is still unclear. Methods CCT3 expression was knocked down by transfecting breast cancer cells with lentiviral shRNA. The proliferation of breast cancer cells (HCC1937 and MDA-MB-231) was detected by Celigo image cytometry and MTT assay, the migration of the cells was measured by Transwell analysis, cell cycle distribution and apoptosis was detected by flow cytometry, and changes in signal transduction proteins were detected by western blot analysis. Results The expression of CCT3 was significantly suppressed by transduction with lentiviral shRNA; CCT3 knockdown significantly reduced the proliferation and metastasis ability of breast cancer cells (HCC 1937 and MDA-MB-231), increased the proportion of cells in S phase, and decreased the proportion of cells in G1 phase compared to those in shControl cells. There was no significant change in the number of cells in the G2/M phase. Apoptosis analysis showed that knockdown of CCT3 induced apoptosis in breast cancer cells. Western blot analysis showed that the expression of many signal transduction proteins was changed after suppression of CCT3. Conclusion CCT3 is closely related to the proliferation and migration of breast cancer and may be a novel therapeutic target.

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