Hypoxic MCF-7 Cells Research Articles

Differential miRNA expression of hypoxic MCF7 and PANC-1 cells.

Hypoxia plays a critical role in the tumor microenvironment by affecting cellular proliferation, metabolism, apoptosis, DNA repair, and chemoresistance. Since hypoxia provokes a distinct shift of microRNA, it is important to illustrate the relative contribution of each hypoxamiR to cancer progression. The present study aims to shed light on the hypoxamiRs that are involved in pancreatic and breast cancer progression to highlight novel targets for the development of new therapies. For 20 cycles, MCF7 breast cancer cells and PANC-1 pancreatic cancer cells were subjected to chronic cyclic hypoxia, which consisted of 72 hours of hypoxia followed by 24 hours of reoxygenation. After 10 and 20 cycles of hypoxia, miRNA expression alterations were profiled using RT-PCR array and further analyzed using a visual analytics platform. The MTT cell proliferation assay was used to determine hypoxic cells' chemoresistance to doxorubicin. Under chronic cyclic hypoxia, hypoxic PANC-1 cells have a comparable doubling time with their normoxic counterparts, whereas hypoxic MCF7 cells show a massive increase in doubling time when compared to their normoxic counterparts. Both hypoxic cell lines developed EMT-like phenotypes as well as doxorubicin resistance. According to the findings of miRNet, 6 and 10 miRNAs were shown to play an important role in enriching six hallmarks of pancreatic cancer in the 10th and 20th cycles of hypoxia, respectively, while 7 and 11 miRNAs were shown to play an important role in enriching the four hallmarks of breast cancer in the 10th and 20th cycles of hypoxia, respectively. miR-221, miR-21, miR-155, and miR-34 were found to be involved in the potentiation of hypoxic PANC-1 hallmarks at both the 10th and 20th cycles, while miR-93, miR-20a, miR-15, and miR-17 were found to be involved in the potentiation of hypoxic MCF7 hallmarks at both the 10th and 20th cycles. This variation in miRNA expression was also connected to the emergence of an EMT-like phenotype, alterations in proliferation rates, and doxorubicin resistance. The chemosensitivity results revealed that chronic cyclic hypoxia is critical in the formation of chemoresistant phenotypes in pancreatic and breast cancer cells. miR-181a and let-7e expression disparities in PANC1, as well as miR-93, miR-34, and miR-27 expression disparities in MCF7, may be associated with the formation of chemoresistant MCF7 and PANC-1 cells following 20 cycles of chronic cyclic hypoxia. Indeed, further research is needed since the particular mechanisms that govern these processes are unknown.

Tyrosol: Repercussion of the Lack of a Hydroxyl-Group in the Response of MCF-7 Cells to Hypoxia.

In solid tumors, such as breast cancer, hypoxic microenvironment worsens patient prognoses. We have previously reported in MCF-7 breast cancer cells that, under hypoxic conditions, hydroxytyrosol (HT) downregulates the level of reactive oxygen species, reduces the expression of hypoxia inducible factor-1α (HIF-1α), and, at high concentrations, can bind to the aryl hydrocarbon receptor (AhR). With this background, the present study investigated whether the most abundant extra virgin olive oil (EVOO) phenolic compound tyrosol (TYR), with a chemical structure similar to HT but with only one hydroxyl group, exerts comparable effects. Our results revealed that, although TYR did not show any antioxidant activity in hypoxic MCF-7 cells, it inhibited the PI3K/Akt/mTOR/S6 kinase (S6K) pathway and reduced the expression of HIF-1α and some of its target genes. Besides, TYR showed a lower binding affinity with the cytosolic transcription factor AhR, and even reduced its transcriptional activity. Some of these results are positive to control tumor progression in a hypoxic environment; however, they are observed at doses unachievable with diet intake or nutraceutical presentations. Considering that EVOO phenols can have synergistic effects, a mixture of low doses of TYR and other phenols could be useful to achieve these beneficial effects.

Modulatory Effect of Vitamin C on Hypoxia Induced Breast Cancer Stem Cells.

Eliminating cancer stem cells (CSCs) is a challenge because of their enhanced resistance to anti-cancer drugs. Vitamin C, which is insufficient in patients with higher stages of cancer, has been gaining attention as a potential treatment for human malignancies. Hence this study aimed to analyze the effect of high-dose vitamin C treatment on the gene expression level of HIF-1α, NF-κB1, BAX, and DNMT1 in the MCF7 cells undergoing hypoxia, as an inducer of CSCs characteristics. As a result, vitamin C could be possibly used as a promising therapeutic adjuvant. Here we first analyzed the breast CSC population alteration in MCF7 cells following hypoxia induction. Then, we evaluated the impact of vitamin C treatment on the gene expression level of four stemness-related genes in hypoxic MCF7 cells. Our results indicate that vitamin C could reduce proliferation and stemness states in CSCs possibly by induction of apoptotic markers such as BAX, along with attenuating stemness markers, including NF-κB1, and DNMT1 gene expressions. According to our findings, vitamin C administration would become a new approach to avoiding the stimulation of CSCs during cancer therapies.

Hypoxia-inducible CircPFKFB4 Promotes Breast Cancer Progression by Facilitating the CRL4DDB2 E3 Ubiquitin Ligase-mediated p27 Degradation

Hypoxic microenvironment and circular RNAs (circRNAs) have shown critical implications in breast cancer (BC) progression. However, the specific functions and underlying mechanisms of circRNAs in BC under hypoxia remain largely unknown. We first screened for differentially expressed circRNAs in normoxic and hypoxic MCF-7 cells using circRNA microarray. A novel hypoxia-induced circRNA, circPFKFB4, was identified. Clinical investigation showed that circPFKFB4 was highly expressed in BC tissues and cell lines, and its overexpression was positively correlated with the advanced clinical stage and poor prognosis of BC patients. Functionally, circPFKFB4 promoted the proliferation of BC cells both in vitro and in vivo. Mechanistically, HIF1α bound to hypoxia response elements in the promoter region of the PFKFB4 gene to facilitate the biogenesis of circPFKFB4 under hypoxia. Hypoxia-induced circPFKFB4 directly bound to both DDB1 and DDB2 and promoted the CRL4DDB2 E3 ubiquitin ligase assembly, resulting in p27 ubiquitination and BC progression under hypoxia. Our findings revealed a novel interaction between circPFKFB4 and the CRL4DDB2 E3 ubiquitin ligase, suggesting that circPFKFB4 might serve as a promising biomarker and therapeutic target for BC.

Label-free detection of hypoxia-induced extracellular vesicle secretion from MCF-7 cells

Nanoscale extracellular vesicles (EVs) including exosomes (50–150 nm membrane particles) have emerged as promising cancer biomarkers due to the carried genetic information about the parental cells. However the sensitive detection of these vesicles remains a challenge. Here we present a label-free electrochemical sensor to measure the EVs secretion levels of hypoxic and normoxic MCF-7 cells. The sensor design includes two consecutive steps; i) Au electrode surface functionalization for anti-CD81 Antibody and ii) EVs capture. The label-free detection of EVs was done via Differential Pulse Voltammetry (DPV) and Electrochemical Impedance Spectroscopy (EIS). The working linear range for the sensor was 102–109 EVs/ml with an LOD 77 EVs/mL and 379 EVs/ml for EIS and DPV based detection. A blood-abundant protein, RhD was used for the selectivity test. In order to assess the performance of the biosensor, the level of EVs secretion by the human breast cancer MCF-7 cell line was compared with enzyme-linked immunosorbent assays (ELISA) and Nanoparticle Tracking Analysis (NTA). Designed label-free electrochemical sensors utilized for quantification of EVs secretion enhancement due to CoCl2-induced hypoxia and 1.23 fold increase with respect to normoxic conditions was found.

The Promoting Effect of Radiation on Glucose Metabolism in Breast Cancer Cells under the Treatment of Cobalt Chloride.

We aimed to investigate the influence of radiation on hypoxia-treated breast cancers cells and its underlying mechanism. We mimicked the hypoxic response in MCF-7 cells by the treatment of CoCl2. Meanwhile, hypoxic MCF-7 cells induced by CoCl2 or untreated MCF-7 cells were treated with or without radiation, and then treated with or without hypoxia inducible factors-1α (HIF-1α) inhibitor. Subsequently, glucose update and lactate release rate were determined by commercial kits, as well as the expressions of HIF-1α and the glucose metabolic pathway related genes, including fructose biphoshatase 1 (FBP1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and isocitrate dehydrogenase 2 (IDH20) were detected by western blotting and/or RT-PCR. The results showed that glucose uptake rate and lactate release rate were increased in cells under hypoxia and/or radiation condition compared with untreated cells (p<0.05), while the addition of HIF-1α inhibitor decreased these rates in hypoxia + radiation treated cells (p<0.05). In addition, compared with untreated cells, the mRNA and protein levels of HIF-1α were significantly increased under hypoxia and radiation condition (p<0.05), while which decreased after the addition of HIF-1α inhibitor (p<0.05). Similar content changing trends (all p<0.05) were observed in FBP1, IDH2, GLUT1, and LDHA but not HK2. In conclusion, the combination of radiation and hypoxia could promote the glucose metabolism. Furthermore, HIF-1α might inhibit the promoting effect of radiation on glycolysis in hypoxic MCF-7 cells by regulating the glucose metabolic pathway.

Carbonic Anhydrase 9 mRNA/microRNA34a Interplay in Hypoxic Human Mammospheres.

The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.

Radiation enhancing effects of sanazole and gemcitabine in hypoxic breast and cervical cancer cells in vitro.

Aim of the studySanazole and gemcitabine have been proven clinically as hypoxic cell radiosensitisers. This study was conducted to determine the radiation enhancing effects of sanazole and gemcitabine when administered together at relevant concentrations into hypoxic human MCF-7 and HeLa cells.Material and methodsA 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to evaluate the number of surviving cells. Cell cycle was determined by flow cytometry. Cell surviving fractions were determined by the standard in vitro colony formation assay.ResultsThe cell colony formation assay indicated that the radiosensitivity of hypoxic MCF-7 and HeLa cells was enhanced by sanazole or gemcitabine. The combination of the two drugs displayed significant radiation enhancing effects at the irradiation doses of 6, 8, and 10 Gy in both cell lines, which were arrested in the S phase.ConclusionsThis study indicated that the co-administration of the two drugs may result in a beneficial gain in radio-therapy for hypoxic breast cancer and cervical cancer.

Synthesis and Evaluation of Polymeric Gold Glyco-Conjugates as Anti-Cancer Agents

The antitumor activity of organo-gold compounds is a focus of research from the past two decades. A variety of gold stabilizing ligands such as vitamins and xanthanes have been prepared and explored for their 'chelating effect' as well as for their antitumor activity. Dithiocarbamates (DTC) compounds and their metallic conjugates have been well explored for their antiproliferative activities. In this study, glycopolymer based DTC-conjugates are prepared by reversible addition-fragmentation chain transfer polymerization (RAFT) and subsequently modified with gold(I) phosphine. These polymer-DTC derivatives and their gold compounds are tested for their in vitro toxicity in both normal and cancer cell lines. The Au(I) phosphine conjugated cationic glycopolymers of 10 kDa and 30 kDa are evaluated for their cytotoxicity profiles using MTT assay. Au(I) compounds are well-known for their mitochondrial toxicity, hence hypoxic cell lines bearing unusually enlarged mitochondria are subjected to these anticancer compounds. It is concluded that these polymeric DTC derivatives and their gold conjugates indeed show higher accumulation as well as cytotoxicity to cancer cells under hypoxic conditions in comparison to the normoxic ones. Hypoxic MCF-7 cells showed significant sensitivity toward the low molecular weight (10 kDa) glycopolymer-Au(I) complexes.

Abstract 3946: Genome-wide characterization of hypoxic breast cancer transcriptome

Abstract Hypoxia is a common feature of most solid tumors and is associated with poor prognosis in breast cancer. In the current study, we investigated the hypoxia transcription landscape of an ER positive breast cancer cell line [MCF7] using high throughput strand-specific total RNA sequencing (tssRNA-seq). MCF7 cells were grown under hypoxia and normoxia (24 hours, 1% O2), and we found about 7.3% of annotated genome got significantly differential expressed in hypoxic stress, which includes 79% (1293) up regulated and 21% (344) down regulated transcripts (P value of &amp;lt;0.01, fold &amp;gt;1.5). Analysis revealed deregulation of a range of non coding transcripts including piwiRNA (0.36%), miRNA (0.098%), sn/snoRNA (0.028%), tRNA (4.5%), lncRNA (6.5%) under hypoxia (P value of &amp;lt;0.01, fold &amp;gt;1.5). Many non-poly A transcripts and natural anti-sense transcripts (n=71) were detected and differently expressed in hypoxia. We characterized two significant up regulated natural anti-sense transcripts in hypoxia and found that these transcripts are long (&amp;gt;200bp), lack of protein coding frames, localized in nucleus and found highly expressed (range fold &amp;gt;5-15) in different hypoxic breast cancer cell lines. To define which hypoxic altered transcripts are dependent on, hypoxia-inducible factor (HIF), we suppressed HIF-alpha subunits in hypoxic MFC-7 cells using short interfering RNA and subjected them to mRNA-seq. Results revealed that HIF-1, HIF-2 and combined HIF-1/2 knockdown resulted in significant down-regulation of 179, 84 and 149 transcripts, respectively, which were up regulated in hypoxic MCF-7 cells. Despite detection of previously reported HIF dependent transcripts, we detected a substantial number of HIF dependent transcripts (n=64) that were not reported before as hypoxia regulated genes. To our understanding, this is first study to show hypoxia dependent natural anti-sense transcripts and genome-wide HIF dependent transcripts analysis at single base pair resolution in hypoxic tumors. These finding will open new avenues in our efforts to understand the hypoxia induced transcriptional response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3946. doi:1538-7445.AM2012-3946

Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor‐dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions

Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia-induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism. The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF-7 cells using Western blot analysis, real-time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF-7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild-type p53 (wt-p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry. Activated RhoA down-regulated p53 protein, which increased VEGF expression in hypoxic MCF-7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin-mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF-7 cells and interacted with each other physically. Furthermore, nutlin-3, a specific MDM2 inhibitor, was capable of reducing activated RhoA-induced p53 protein stability and attenuating VEGF accumulation. In an MCF-7-HUVEC coculture system, nutlin-3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt-p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt-p53 expression, and high VEGF expression. The current data suggested that activated RhoA promotes VEGF expression and hypoxia-induced angiogenesis through the up-regulation of MDM2 to decrease p53 stability.

Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism

BackgroundSome breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines.MethodsMigration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested.ResultsHypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration.ConclusionsHypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients.

Involvement of AMPK/mTOR/HIF-1α in anticancer control of quercetin in hypoxic MCF-7 cells

Quercetin has been reported to possess therapeutic effects in the treatment of cancers. In this study, the molecular action of quercetin, with emphasis on its ability to control the intracellular signaling cascades of hypoxia inducible factor-1α (HIF-1α), mammalian target of rapamycin (mTOR), and AMP-activated protein kinase (AMPK), responsible for survival or induction of apoptosis in hypoxic MCF-7 cells, was investigated. The effects of quercetin on apoptosis in relation to its ability to prevent HIF-1α induction were investigated. The involvement of HIF-1α reduction in quercetin-based cancer control was clearly shown in conditions of mTOR inhibition by rapamycin, an mTOR inhibitor. Surprisingly, quercetin induced an AMPK-suppressed state in a CoCl2-induced hypoxic condition. It is speculated that quercetin is capable of inhibiting AMPK to decrease HIF-1α, which is a critical survival factor in hypoxia. A complex control of HIF-1α, mTOR, and AMPK is necessary in inducing apoptosis of MCF-7 breast cancer cells under hypoxia.

T1191 Intra-Tumoral Variability in Prostaglandin E 2 Levels in Human Colorectal Cancer Liver Metastases is Associated With Differences in NAD+ Levels and Expression of NAD+-Dependent 15-Prostaglandin Dehydrogenase

Introduction Cyclooxygenase (COX)-2 (synthesis) and NAD+-dependent 15-prostaglandin (PG) dehydrogenase (15-PGDH; catabolism) are rate-limiting enzymes in control of tissue PGE2 levels. COX-2 and 15-PGDH function may be affected by the tumour micro-environment (eg, hypoxia) leading to changes in local PGE2 concentration and altered cancer cell behaviour. Epithelial-mesenchymal transition (EMT) is believed to be crucial for tumour cell invasion/metastasis. EMT is promoted by PGE2 in several cell types in vitro. We investigated the relationship between PGE2 levels, COX-2 and 15-PGDH expression, and NAD+ levels in human colorectal cancer liver metastases (CRCLM), and the role of hypoxia and PGE2 in EMT of CRC cells in vitro. Methods PGE2 and NAD+ levels, and immunoreactive 15-PGDH and COX-2 protein content, were measured in paired central and peripheral tissue from 20 CRCLM. 15-PGDH activity was measured by a [ 3 H]-PGE2 degradation assay in the presence or absence of exogenous NAD+ (1 μM). Parallel in vitro studies used a LIM1863 human CRC cell model of transforming growth factor (TGF) β-induced EMT and MCF-7 human breast cancer cells. Results PGE2 levels were increased by a mean (95% CI) of 26% (1% to 52%) in the centre of CRCLM relative to peripheral tissue (mean 762 vs 604 pg/mg). Counter-intuitively, immunoreactive 15-PGDH protein levels were also increased (14% (3% to 27%)) in tumour centres, as was 15-PGDH activity in the presence of added NAD+ (16% (3% to 29%)). However, NAD+ levels were 59% (25% to 72%) lower in the centre of CRCLM than the periphery (mean 192 vs 546 pmol/mg) suggesting inefficient 15-PGDH-dependent catabolism may explain raised PGE2 levels. By contrast, there were no significant regional differences in immunoreactive COX-2 protein levels. In central areas of CRCLM, cancer cells exhibiting low E-cadherin expression had higher 15-PGDH expression than neighbouring E-cadherin-positive cells. Our CRCLM tissue observations were mirrored by LIM1863 cells undergoing EMT as hypoxic (1% O2) LIM1863 cells contained significantly less NAD+ and exhibited higher 15-PGDH protein levels than normoxic cells. 15-PGDH activity was significantly decreased in hypoxic MCF-7 cells compared with normoxic counterparts when NAD+ was present at a limiting concentration (no exogenous added). Furthermore, PGE2 (0.1–10 μM) promoted TGF-β-induced EMT in LIM 1863 cells in a concentration-dependent manner. Conclusion Regional differences in PGE2 levels and 15-PGDH expression/activity are apparent in human CRCLM (possibly due to a hypoxic micro-environment). PGE2 promotes TGF-β-induced EMT in human CRC cells in vitro. Modulation of factors controlling PGE2 represents a novel therapeutic strategy against EMT in CRCLM.

PWE-070 Intratumoural variability in prostaglandin E2 levels in human colorectal cancer liver metastases is associated with differences in NAD+ levels and expression of NAD+-dependent 15-prostaglandin dehydrogenase

<h3>Introduction</h3> Cyclooxygenase (COX)-2 (synthesis) and NAD+-dependent 15-prostaglandin (PG) dehydrogenase (15-PGDH; catabolism) are rate-limiting enzymes in control of tissue PGE2 levels. COX-2 and 15-PGDH function may be affected by the tumour micro-environment (eg, hypoxia) leading to changes in local PGE2 concentration and altered cancer cell behaviour. Epithelial-mesenchymal transition (EMT) is believed to be crucial for tumour cell invasion/metastasis. EMT is promoted by PGE2 in several cell types in vitro. We investigated the relationship between PGE2 levels, COX-2 and 15-PGDH expression, and NAD+ levels in human colorectal cancer liver metastases (CRCLM), and the role of hypoxia and PGE2 in EMT of CRC cells in vitro. <h3>Methods</h3> PGE2 and NAD+ levels, and immunoreactive 15-PGDH and COX-2 protein content, were measured in paired central and peripheral tissue from 20 CRCLM. 15-PGDH activity was measured by a [³H]-PGE2 degradation assay in the presence or absence of exogenous NAD+ (1 μM). Parallel in vitro studies used a LIM1863 human CRC cell model of transforming growth factor (TGF) β-induced EMT and MCF-7 human breast cancer cells. <h3>Results</h3> PGE2 levels were increased by a mean (95% CI) of 26% (1% to 52%) in the centre of CRCLM relative to peripheral tissue (mean 762 vs 604 pg/mg). Counter-intuitively, immunoreactive 15-PGDH protein levels were also increased (14% (3% to 27%)) in tumour centres, as was 15-PGDH activity in the presence of added NAD+ (16% (3% to 29%)). However, NAD+ levels were 59% (25% to 72%) lower in the centre of CRCLM than the periphery (mean 192 vs 546 pmol/mg) suggesting inefficient 15-PGDH-dependent catabolism may explain raised PGE2 levels. By contrast, there were no significant regional differences in immunoreactive COX-2 protein levels. In central areas of CRCLM, cancer cells exhibiting low E-cadherin expression had higher 15-PGDH expression than neighbouring E-cadherin-positive cells. Our CRCLM tissue observations were mirrored by LIM1863 cells undergoing EMT as hypoxic (1% O2) LIM1863 cells contained significantly less NAD+ and exhibited higher 15-PGDH protein levels than normoxic cells. 15-PGDH activity was significantly decreased in hypoxic MCF-7 cells compared with normoxic counterparts when NAD+ was present at a limiting concentration (no exogenous added). Furthermore, PGE2 (0.1–10 μM) promoted TGF-β-induced EMT in LIM 1863 cells in a concentration-dependent manner. <h3>Conclusion</h3> Regional differences in PGE2 levels and 15-PGDH expression/activity are apparent in human CRCLM (possibly due to a hypoxic micro-environment). PGE2 promotes TGF-β-induced EMT in human CRC cells in vitro. Modulation of factors controlling PGE2 represents a novel therapeutic strategy against EMT in CRCLM.

Up‐regulation of pro‐inflammatory genes as adaptation to hypoxia in MCF‐7 cells and in human mammary invasive carcinoma microenvironment

The role of tumor cells in synthesizing pro-inflammatory molecules is still controversial. Here we report that hypoxic treatment of the MCF-7 human mammary adenocarcinoma cell line induced activation of hypoxia-inducible factor 1alpha (HIF-1alpha) and nuclear factor-kappa B (NF-kappaB). Importantly, hypoxia regulated expression of alarmin receptors such as the receptor for advanced glycation end products (RAGE) and the purinoreceptor (P2X7R), and up-regulated inflammatory response (IR) genes such as the inducible enzymes nitric oxide synthase (NOS2), cycloxygenase (COX2), and the acute-phase protein pentraxin-3 (PTX3). Hypoxia also stimulated chemokine (C-X-C motif) receptor 4 (CXCR4) mRNA synthesis. In fact, the CXCR4 ligand stromal-derived factor-1alpha (SDF-1alpha) increased invasion and migration of hypoxic MCF-7 cells. Inhibition of HIF-1alpha by chetomin and NF-kappaB by parthenolide reduced mRNA and protein expression of the studied molecules and prevented invasion of hypoxic MCF-7 cells. Moreover, solid invasive mammary tumor microenvironment was analyzed after laser-capture microdissection (LCMD) comparing tumor versus host normal tissue. Nuclear translocation of HIF-1alpha and NF-kappaB and up-regulation of IR, CXCR4, estrogen receptor alpha (ERalpha), and epithelial growth factor receptor (EGFR) was observed in tumor but not in host normal tissue in the absence of a local inflammatory leukocyte infiltrate. We conclude that under hypoxic conditions MCF-7 cells acquire a pro-inflammatory phenotype, and that solid human mammary carcinoma evidenced a similar activation of HIF-1alpha, NF-kappaB, and IR genes in malignant tumor cells as compared to the normal host tissues. We suggest a role for IR activation in the malignant progression of transformed cells.

Radiobiological effects of hypoxia-dependent uptake of 64Cu-ATSM: enhanced DNA damage and cytotoxicity in hypoxic cells

Hypoxia occurs frequently in cancers and can lead to therapeutic resistance due to poor perfusion and loss of the oxygen enhancement effect. (64)Cu-ATSM has shown promise as a hypoxia diagnostic agent due to its selective uptake and retention in hypoxic cells and its emission of positrons for PET imaging. (64)Cu also emits radiotoxic Auger electrons and beta(-) particles and may therefore exhibit therapeutic potential when concentrated in hypoxic tissue. MCF-7 cells were treated with 0-10 MBq/ml (64)Cu-ATSM under differing oxygen conditions ranging from normoxia to severe hypoxia. Intracellular response to hypoxia was measured using Western blotting for expression of HIF-1alpha, while cellular accumulation of (64)Cu was measured by gamma counting. DNA damage and cytotoxicity were measured with, respectively, the Comet assay and clonogenic survival. (64)Cu-ATSM uptake in MCF-7 cells increased as atmospheric oxygen decreased (up to 5.6 Bq/cell at 20.9% oxygen, 10.4 Bq/cell at 0.1% oxygen and 26.0 Bq/cell at anoxia). Toxicity of (64)Cu-ATSM in MCF-7 cells also increased as atmospheric oxygen decreased, with survival of 9.8, 1.5 and 0% in cells exposed to 10 MBq/ml at 20.9, 0.1 and 0% oxygen. The Comet assay revealed a statistically significant increase in (64)Cu-ATSM-induced DNA damage under hypoxic conditions. The results support a model in which hypoxia-enhanced uptake of radiotoxic (64)Cu induces sufficient DNA damage and toxicity to overcome the documented radioresistance in hypoxic MCF-7 cells. This suggests that (64)Cu-ATSM and related complexes have potential for targeted radionuclide therapy of hypoxic tumours.

Hypoxia enhances the replication of oncolytic herpes simplex virus in p53- breast cancer cells

Hypoxic cancer cells are refractory to conventional chemotherapy. Herpes simplex virus type-1 (HSV-1)-derived oncolytic viruses are safe for therapy since they lack the neurovirulence gene ICP34.5. Cancer cells containing high MEK activity are permissive to the HSV-1-derived oncolytic virus, R3616. Considering that hypoxia increases MEK activity, we determined whether hypoxic MDA-MB-231 and MCF-7 cells were more permissive to R3616, compared to normoxic cells. We observed nine-fold higher (3.5 x 10e6 pfu/ 4 x 10e5 pfu/ml) titers in MDA-MB-231 hypoxic cells compared to normoxic; however, hypoxic MCF-7 cells did not yield higher R3616 titers. Markers for early and late viral infection were consistent with this result: 1) virus-induced chaperone-enriched (VICE) domains were observed in MDA-MB-231 cells, and 2) the HSV-1 glycoprotein C (gC), a protein produced late in infection, accumulated in hypoxic MDA-MB-231 cells. Thus, oncolytic R3616 virus may target hypoxic p53- breast cancer cells.

Cytotoxicity of Paclitaxel Incorporated in PLGA Nanoparticles on Hypoxic Human Tumor Cells

The aim of this work was to prepare paclitaxel-loaded PLGA nanoparticles and determine cytotoxicity of released paclitaxel for two hypoxic human tumor cell lines: breast carcinoma (MCF-7) and carcinoma cervicis (HeLa). Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. Physicochemical characteristics of nanoparticles were studied. Cellular uptake of nanoparticles was evaluated by transmission electronic microscopy and fluorescence microscopy. Flow cytometry quantified the number of cells held in G(2)/M phase. Cell viability was determined by the ability of single cell to form colonies. Biodistribution of nanoparticles in mice was evaluated by fluorescence microscopy. The nanoparticles were spherical with average diameter 318 +/- 5.1 nm. The encapsulation efficiency was 88.52%. The drug release profile in vitro exhibited a biphasic pattern. Cellular uptake was observed. Co-culture of tumor cells with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in G(2)/M phase. Paclitaxel-loaded nanoparticles exhibited cytotoxic effect on both hypoxic MCF-7 and HeLa cells and its cytotoxicity was more significant than that of free paclitaxel. Fluorescent nanoparticles were mainly distributed to liver and spleen of mice. Paclitaxel-loaded PLGA nanoparticles may be considered a promising drug delivery system to eradicate hypoxic tumor cells.

The effect of paclitaxel-loaded nanoparticles with radiation on hypoxic MCF-7 cells

The inability of radiotherapy to eradicate completely certain human tumours may be due to the presence of resistant hypoxic cells. Several studies have confirmed the radiosensitizing effect of paclitaxel, a microtubular inhibitor. The object of this study was to evaluate the physicochemical characteristics of paclitaxel-loaded nanoparticles, and determine the ability of the released paclitaxel to radiosensitize hypoxic human breast carcinoma cells (MCF-7) with respect to radiation dose. The poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. The morphology of the paclitaxel-loaded nanoparticles was investigated by scanning electron microscopy. The drug encapsulation efficiency (EE) and in vitro release profile were measured by high-performance liquid chromatography. Cell cycle was evaluated by flow cytometry. Cell viability was measured by the ability of single cells to form colonies in vitro. The prepared nanoparticles were spherical with diameter between 200 and 800 nm. The EE was 85.5%. The drug release pattern was biphasic with a fast release rate followed by a slow one. Co-culture of human breast carcinoma cells (MCF-7) with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in the G2/M phase of the cell cycle and effectively sensitized hypoxic MCF-7 cells to radiation with radiosensitivity shown to be dependent of radiation dose at levels of dosages studied. The sensitizer enhancement ratio for paclitaxe-loaded nanoparticles at 10% survival is approximately 1.4. This work has demonstrated that paclitaxel can be effectively released from a biodegradable PLGA nanoparticle delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities for hypoxic tumour cells.