scholarly journals Broussochalcone A Induces Apoptosis in Human Renal Cancer Cells via ROS Level Elevation and Activation of FOXO3 Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Han Ki Lee ◽  
Hyo Sun Cha ◽  
Myeong Jin Nam ◽  
Kyungmoon Park ◽  
Yung-Hun Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Renal Cancer ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Apoptotic Effects

Broussochalcone A (BCA) is a chalcone compound extracted from the cortex of Broussonetiapapyrifera (L.) Ventenat that exerts various effects, such as potent antioxidant, antiplatelet, and anticancer effects. However, the effects of BCA against cancers have been seldom studied. This study is aimed at demonstrating the apoptotic mechanisms of BCA in A498 and ACHN cells, which are two types of human renal cancer cell lines. MTT, cell counting, and colony formation assays indicated that BCA treatment inhibited cell viability and cell growth. Further, cell cycle analysis revealed that BCA induced cell cycle arrest at the G2/M phase. Annexin V/PI staining and TUNEL assays were performed to determine the apoptotic effects and DNA fragmentation after treatment with BCA. Based on western blot analysis, BCA induced the upregulation of cleaved PARP, FOXO3, Bax, p21, p27, p53, phosphorylated p53 (ser15, ser20, and ser46), and active forms of caspase-3, caspase-7, and caspase-9 proteins, but downregulated the proforms of the proteins. The expression levels of pAkt, Bcl-2, and Bcl-xL were also found to be downregulated. Western blot analysis of nuclear fractionation results revealed that BCA induced the nuclear translocation of FOXO3, which might be induced by DNA damage owing to the accumulation of reactive oxygen species (ROS). Elevated intracellular ROS levels were also found following BCA treatment. Furthermore, DNA damage was detected after BCA treatment using a comet assay. The purpose of this study was to elucidate the apoptotic effects of BCA against renal cancer A498 and ACHN cells. Collectively, our study findings revealed that the apoptotic effects of BCA against human renal cancer cells occur via the elevation of ROS level and activation of the FOXO3 signaling pathway.

Neferine-induced apoptosis is dependent on the suppression of Bcl-2 expression via downregulation of p65 in renal cancer cells

2019 ◽  
Vol 51 (7) ◽  
pp. 734-742 ◽  
Author(s):  
Eun-Ae Kim ◽  
Eon-Gi Sung ◽  
In-Hwan Song ◽  
Joo-Young Kim ◽  
Hwa-Jung Sung ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Renal Cancer ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Induced Apoptosis

Abstract Neferine is an alkaloid extracted from a seed embryo of Nelumbo nucifera and has recently been shown to have anticancer effects in various human cancer cell lines. However, the detailed molecular mechanism of neferine-induced apoptosis has not been elucidated in renal cancer cells. In the present study, we observed that neferine induced inhibition of cell proliferation and apoptosis in Caki-1 cells in a dose-dependent manner by using MT assay and flow cytometry and that neferine-mediated apoptosis was attenuated by pretreatment with N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone, a pan-caspase inhibitor. Treatments with neferine dose-dependently downregulated B cell lymphoma-2 (Bcl-2) expression at the transcriptional level determined by reverse transcriptase-polymerase chain reaction. The forced expression of Bcl-2 and p65 attenuated the neferine-mediated apoptosis in Caki-1 cells. In addition, neferine induced apoptosis by downregulating Bcl-2 and p65 expression in the other two kidney cancer cell lines determined by flow cytometry and western blot analysis. Finally, we observed that treatment with neferine induced apoptosis by inhibiting the NF-κB pathway through caspase-mediated cleavage of the p65 protein by western blot analysis. Collectively, this study demonstrated that neferine-induced apoptosis is mediated by the downregulation of Bcl-2 expression via repression of the NF-κB pathway in renal cancer cells.

Carboplatin-induced Senescence in Ovarian Cancer Cells Is Reversed Through EGFR and NF-κB Signaling

2021 ◽  
Author(s):  
Yue Li ◽  
Mingxu Fu ◽  
Ling Guo ◽  
Xiaoxiao Sun ◽  
Yuhang Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Blue Staining ◽  
Ovarian Cancer Cells ◽  
Positive Staining ◽  
The Impact

Abstract Background: Metastases and recurrence of ovarian cancer after surgery and chemotherapy account for most cancer-related deaths, yet the mechanism underlying metastases and recurrence remains poorly understood. Recent evidence demonstrates that although long-lasting cells were considered tumor suppressors, senescent cancer cells, can induce the metastases and recurrence. In this study, we focused on the fate of ovarian cancer cells treated with carboplatin and explored the mechanism underlying ovarian cancer cell recovery from chemotherapy-induced senescence. Methods: SÁ-β-galactosidase staining was used to detect the impact of carboplatin on senescence of ovarian cancer cells. Cell proliferation was determined using direct cell counting, clone formation assay and 3D tumor spheroid formation assay. Lentivirus-mediated transduction was used to silence or upregulate EGFR expression. Quantitative real-time PCR and western blot analysis validated the efficacy of the knockdown or overexpression effect. Immunofluorescence staining and western blot analysis were used to examined the expression of EGFR and NF-KB. Cell death was determined using trypan blue staining assay. Results: Ovarian cancer cells treated by carboplatin exhibit a senescence-like phenotype indicated by SA-β-galactosidase positive staining. Importantly, carboplatin-induced senescence-like phenotype is reversible. In ovarian cancer cells, EGFR positively regulated cells proliferation, decreased carboplatin-induced senescence and upregulated the NF-κB1 protein level. EGFR/NF-κB1 upregulation promoted the recovery of ovarian cancer cells from senescence and chemoresistance to carboplatin. Conclusions: Ovarian cancer cells treated with carboplatin displayed a reversible senescence-like phenotype that could be combined with EGFR or NF-κB1 inhibitors to improve treatment effects.

scholarly journals Critical Roles of the PI3K-Akt-mTOR Signaling Pathway in Apoptosis and Autophagy of Astrocytes Induced by Methamphetamine

2019 ◽  
Vol 17 (1) ◽  
pp. 96-104 ◽  
Author(s):  
Han-Qing Liu ◽  
Ya-Wen An ◽  
A-Zhen Hu ◽  
Ming-Hua Li ◽  
Jue-Lian Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Cell Counting ◽  
Mtor Signaling Pathway ◽  
Induced Apoptosis

AbstractThis study aimed to reveal potential roles of the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in apoptosis and autophagy of astrocytes induced by methamphetamine (METH). A Cell Counting Kit-8 (CCK-8) was used to determine the reduction in proliferation of U-118 MG cells induced by METH. Hoechst 33258 and flow cytometry were used to observe the astrocytes. Western blot analysis was performed to evaluate protein expression and phosphorylation levels. METH inhibited the proliferation of U-118 MG cells and induced apoptosis and autophagy. Western blot analysis showed that the ratio of LC3-II/I was increased, whereas the expression of Bcl-2 was decreased. The phosphorylation cascade of kinases in the PI3K-Akt-mTOR signaling pathway was significantly inhibited by METH exposure, as were proteins downstream of mTORC1, such as p70s6k, rps6, 4EBP1 and eIF4E. METH inhibited proliferation of U-118 MG cells and induced apoptosis and autophagy. The PI3K-Akt-mTOR signaling pathway likely plays a critical role in these effects.

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 Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

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.

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.

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.

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