Characterization of choline kinase in human endothelial cells

High choline kinase‐α (Chk‐α) expression is frequently observed in cancer cells, making it a novel target for pharmacological and molecular inhibition. As inhibiting agents are delivered systemically, it is important to determine Chk‐α expression levels in endothelial cells that line both normal and tumor vasculature, and the effect of Chk‐α downregulation on these cells. Here, we characterized Chk‐α expression and the effect of its downregulation in human umbilical vein endothelial cells (HUVECs) relative to MDA‐MB‐231 human breast cancer cells. We used small interfering RNA (siRNA) to downregulate Chk‐α expression. Basal mRNA levels of Chk‐α were approximately three‐fold lower in HUVECs relative to MDA‐MB‐231 breast cancer cells. Consistent with the differences in Chk‐α protein levels, phosphocholine levels were approximately 10‐fold lower in HUVECs relative to MDA‐MB‐231 cells. Transient transfection with siRNA‐Chk resulted in comparable levels of mRNA and protein in MDA‐MB‐231 breast cancer cells and HUVECs. However, there was a significant reduction in proliferation in MDA‐MB‐231 cells, but not in HUVECs. No significant difference in CD31 immunostaining was observed in tumor sections obtained from mice injected with control luciferase‐short hairpin (sh)RNA or Chk‐shRNA lentivirus. These data suggest that systemically delivered agents that downregulate Chk‐α in tumors will not affect endothelial cell proliferation during delivery, and further support the development of Chk‐α downregulation as a cancer‐specific treatment. Copyright © 2013 John Wiley & Sons, Ltd.     

Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells

A direct correlation exists between increased choline kinase (Chk) expression, and the resulting increase of phosphocholine levels, and histological tumor grade. To better understand the function of Chk and choline phospholipid metabolism in breast cancer we have stably overexpressed one of the two isoforms of Chk‐α known to be upregulated in malignant cells, in non‐invasive MCF‐7 human breast cancer cells. Dynamic tracking of cell invasion and cell metabolism were studied with a magnetic resonance (MR) compatible cell perfusion assay. The MR based invasion assay demonstrated that MCF‐7 cells overexpressing Chk‐α (MCF‐7‐Chk) exhibited an increase of invasion relative to control MCF‐7 cells (0.84 vs 0.3). Proton MR spectroscopy studies showed significantly higher phosphocholine and elevated triglyceride signals in Chk overexpressing clones compared to control cells. A test of drug resistance in MCF‐7‐Chk cells revealed that these cells had an increased resistance to 5‐fluorouracil and higher expression of thymidylate synthase compared to control MCF‐7 cells. To further characterize increased drug resistance in these cells, we performed rhodamine‐123 efflux studies to evaluate drug efflux pumps. MCF‐7‐Chk cells effluxed twice as much rhodamine‐123 compared to MCF‐7 cells. Chk‐α overexpression resulted in MCF‐7 human breast cancer cells acquiring an increasingly aggressive phenotype, supporting the role of Chk‐α in mediating invasion and drug resistance, and the use of phosphocholine as a biomarker of aggressive breast cancers. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular causes of elevated phosphoethanolamine in breast and pancreatic cancer cells

Elevated phosphoethanolamine (PE) is frequently observed in MRS studies of human cancers and xenografts. The role of PE in cell survival and the molecular causes underlying this increase are, however, relatively underexplored. In this study, we investigated the roles of ethanolamine kinases (Etnk‐1 and 2) and choline kinases (Chk‐α and β) in contributing to increased PE in human breast and pancreatic cancer cells. We investigated the effect of silencing Etnk‐1 and Etnk‐2 on cell viability as a potential therapeutic strategy. Both breast and pancreatic cancer cells showed higher PE compared with their nonmalignant counterparts. We identified Etnk‐1 as a major cause of the elevated PE levels in these cancer cells, with little or no contribution from Chk‐α, Chk‐β, or Etnk‐2. The increase of PE observed in pancreatic cancer cells in culture was replicated in the corresponding tumor xenografts. Downregulation of Etnk‐1 with siRNA resulted in cell cytotoxicity that correlated with PE levels in breast and pancreatic cancer cells. Etnk‐1 may provide a potential therapeutic target in breast and pancreatic cancers.     

Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation,migration, and invasion

Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF‐7 and MDA‐MB‐231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC‐specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF‐7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA‐MB‐231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of breast cancers and therapy response by MRS and quantitative gene expression profiling in the choline pathway

Tumor choline metabolites have potential for use as diagnostic indicators of breast cancer phenotype and can be non‐invasively monitored in vivo by MRS. Extract studies have determined that the principle diagnostic component of these peaks is phosphocholine (PCho), the biosynthetic precursor to the membrane phospholipid, phosphatidylcholine (PtdCho). The ability to resolve and quantify PCho in vivo would improve the accuracy of this putative diagnostic tool. In addition, determining the biochemical mechanisms underlying these metabolic perturbations will improve the understanding of breast cancer and may suggest potential molecular targets for drug development. Reported herein is the in vivo resolution and quantification of PCho and glycerophosphocholine (GPC) in breast cancer xenografts in SCID mice via image‐guided ³¹P MRS, localized to a single voxel. Tumor metabolites are also detected using ex vivo extracts and high‐resolution NMR spectroscopy and are quantified in the metastatic tumor line, MDA‐mb‐231. Also reported is the quantification of cytosolic and lipid metabolites in breast cells of differing cancer phenotype, and the identification of metabolites that differ among these cell lines. In cell extracts, PCho and the PtdCho breakdown products, lysophosphatidylcholine, GPC and glycerol 3‐phosphate, are all raised in breast cancer lines relative to an immortalized non‐malignant line. These metabolic differences are in direct agreement with differences in expression of genes encoding enzymes in the choline metabolic pathway. Results of this study are consistent with previous studies, which have concluded that increased choline uptake, increased choline kinase activity, and increased phosholipase‐mediated turnover of PtdCho contribute to the observed increase in PCho in breast cancer. In addition, this study presents evidence suggesting a specific role for phospholipase A₂‐mediated PtdCho catabolism. Gene expression changes following taxane therapy are also reported and are consistent with previously reported changes in choline metabolites after the same therapy in the same tumor model. Copyright © 2008 John Wiley & Sons, Ltd.     

Silencing of the glycerophosphocholine phosphodiesterase GDPD5 alters the phospholipid metabolite profile in a breast cancer model in vivo as monitored by 31P MRS

Abnormal choline phospholipid metabolism is an emerging hallmark of cancer, which is implicated in carcinogenesis and tumor progression. The malignant metabolic phenotype is characterized by high levels of phosphocholine (PC) and relatively low levels of glycerophosphocholine (GPC) in aggressive breast cancer cells. Phosphorus (³¹P) MRS is able to non‐invasively detect these water‐soluble metabolites of choline as well as ethanolamine phospholipid metabolism. Here we have investigated the effects of stably silencing glycerophosphoester diesterase domain containing 5 (GDPD5), which is an enzyme with glycerophosphocholine phosphodiesterase activity, in MDA‐MB‐231 breast cancer cells and orthotopic tumor xenografts. Tumors in which GDPD5 was stably silenced with GDPD5‐specific shRNA contained increased levels of GPC and phosphoethanolamine (PE) compared with control tumors. Copyright © 2014 John Wiley & Sons, Ltd.     

HDAC1调控Wnt/β-catenin信号通路对乳腺癌细胞凋亡的影响

目的:研究组蛋白脱乙酰酶1(HDAC1)对乳腺癌细胞凋亡的影响及机制。方法:RT-qPCR和Western blot法分别测定正常乳腺上皮细胞系MCF-10A和乳腺癌细胞系BT549、MCF-7和MDA-MB-231中HDAC1的m RNA和蛋白水平。在MDA-MB-231细胞中转染HDAC1 si RNA,RT-qPCR和Western blot测定HDAC1的表达水平,MTT法测定细胞活力,流式细胞术测定凋亡,Western blot测定细胞中β-连环蛋白(β-catenin)、c-Myc、细胞周期蛋白D1(cyclin D1)和cleaved caspase-3的蛋白水平。用Wnt/β-catenin信号通路激活剂处理下调HDAC1表达后的乳腺癌细胞,测定细胞活力和凋亡。结果:乳腺癌细胞系BT549、MCF-7和MDA-MB-231中HDAC1的m RNA和蛋白水平均明显高于正常乳腺上皮细胞系MCF-10A(P 0. 01),并且MDA-MB-231细胞中的HDAC1水平最高。HDAC1 si RNA可以降低乳腺癌细胞中HDAC1的m RNA和蛋白水平。敲减HDAC1表达后的MDA-MB-231细胞活力降低,细胞凋亡率升高,细胞中cleaved caspase-3水平升高,β-catenin、c-Myc和cyclin D1的蛋白水平降低(P 0. 05)。Wnt/β-catenin信号通路激活剂可以逆转HDAC1下调诱导的MDA-MB-231细胞凋亡和细胞活力降低,减少cleaved caspase-3的水平(P 0. 05)。结论:敲减HDAC1的表达可以通过抑制Wnt/β-catenin信号通路的激活诱导乳腺癌细胞凋亡。     

Oestrogen‐mediated phosphorylation and stabilization of BRCA2 protein in breast

Disease‐associated BRCA2 mutations typically result in protein truncations that delete the phosphorylation‐regulated S3291 BRCA2 domain that interacts with Rad51. BRCA2 hereditary breast cancers are usually ER⁺, differing from BRCA1 hereditary cancers, which are usually ER^?. We studied BRCA2 protein expression and S3291 phosphorylation in normal breast tissues and in sporadic breast cancers and observed that BRCA2 is expressed and phosphorylated in normal breast and 10 ER⁺ breast cancers but not in 10 ER^? breast cancers. In order to study this correlation between ER and BRCA2 expression, we studied ER⁺ breast cancer cell lines. We found that a rapid increase in BRCA2 S3291 phosphorylation occurs following 17‐β‐oestradiol (E2) treatment. This increase seen in BRCA2 total and phospho‐S3291 protein levels was found to be unaffected with cycloheximide pre‐treatment, but decreased following tamoxifen, ICI 182,780 or roscovitine treatment. This suggests a requirement for ER and cdk (cyclin‐dependent kinase) in mediating the increased protein levels. MCF7 cell cycle distribution analysis following E2, in both the presence and absence of roscovitine (a cdk inhibitor), did not demonstrate any changes during an 8 h period, which further supports our hypothesis that mitogenic effects of E2 are not predominant at early time points. Studies with MG132 proteasome inhibitor and siRNA to skp2 support a model in which skp2‐mediated proteasomal degradation of BRCA2 rapidly degrades BRCA2 protein in the absence of hormone treatment, which likely inhibits this pathway. E2 was shown to improve survival of MCF7 cells upon radiation treatment and roscovitine partially reversed this effect. We have demonstrated that BRCA2 protein is specifically expressed in ER⁺ breast cancers and are investigating a pathway that may show a link between E2 action and BRCA2 protein function in breast cancer. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MR‐detectable metabolic consequences of mitogen‐activated protein kinase kinase (MEK) inhibition

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS‐detectable metabolic consequences of mitogen‐activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF‐7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS‐detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF‐7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized ¹³C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3‐kinase pathway and AMP‐activated protein kinase. Our findings demonstrate the value of translatable non‐invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition. Copyright © 2014 John Wiley & Sons, Ltd.     

Noninvasive imaging identifies new roles for cyclooxygenase-2 in choline and lipid metabolism of human breast cancer cells

The expression of cyclooxygenase-2 (COX-2) is observed in approximately 40% of breast cancers. A major product of the COX-2-catalyzed reaction, prostaglandin E(2), is an inflammatory mediator that participates in several biological processes, and influences invasion, vascularization and metastasis. Using noninvasive MRI and MRS, we determined the effect of COX-2 downregulation on the metabolism and invasion of intact poorly differentiated MDA-MB-231 human breast cancer cells stably expressing COX-2 short hairpin RNA. Dynamic tracking of invasion, extracellular matrix degradation and metabolism was performed with an MRI- and MRS-compatible cell perfusion assay under controlled conditions of pH, temperature and oxygenation over the course of 48 h. COX-2-silenced cells exhibited a significant decrease in invasion relative to parental cells that was consistent with the reduced expression of invasion-associated matrix metalloproteinase genes and an increased level of the tissue inhibitor of metalloproteinase-1. We identified, for the first time, a role for COX-2 in mediating changes in choline phospholipid metabolism, and established that choline kinase expression is partly dependent on COX-2 function. COX-2 silencing resulted in a significant decrease in phosphocholine and total choline that was detected by MRS. In addition, a significant increase in lipids, as well as lipid droplet formation, was observed. COX-2 silencing transformed parental cell metabolite patterns to those characteristic of less aggressive cancer cells. These new functional roles of COX-2 may identify new biomarkers and new targets for use in combination with COX-2 targeting to prevent invasion and metastasis.     

干扰Yes相关蛋白表达调控Wnt/β-catenin信号通路影响膀胱癌细胞凋亡

目的:探讨干扰Yes相关蛋白(YAP)的表达对膀胱癌细胞凋亡的影响及机制。方法:以膀胱癌细胞T-24为研究对象,分为正常对照(control)组、siRNA阴性对照(siRNA-NC)组和YAP siRNA组。分别以real-time PCR和Western blot实验检测转染后各组细胞中YAP的mRNA和蛋白表达水平,MTT法检测细胞活力,流式细胞术检测细胞凋亡情况,Western blot检测β-连环蛋白(β-catenin)和c-Myc表达水平。用Wnt/β-catenin信号通路抑制剂FH535处理YAP siRNA组细胞,并用MTT法检测细胞活力,流式细胞术检测细胞凋亡。结果:YAP siRNA组YAP的mRNA和蛋白水平均明显低于control组(P0.05)。YAP siRNA组的细胞活力及c-Myc和β-catenin水平均明显低于control组(P0.05),而细胞凋亡率明显高于control组(P0.05)。与未经FH535处理的YAP siRNA组细胞相比,YAP siRNA组的细胞经FH535处理后细胞活力明显下降,细胞凋亡率升高,差异有统计学意义(P0.05)。结论:干扰YAP表达能够通过抑制Wnt/β-catenin信号通路激活而抑制膀胱癌细胞活力,并促进膀胱癌细胞凋亡。     

Small interfering RNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cell invasion, survival, and tumorigenicity

A major cause of tumor treatment failure is cancer cell metastasis. Toll-like receptor 4 (TLR4)-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. In this study, we investigated the biological roles of TLR4 in prostate metastatic cell invasion and survival, and the potential of gene silencing of TLR4 using small interfering RNA (siRNA) for treatment of cancer. In cultured human prostate cancer cell lines, TLR4 were higher PC3 and DU145 as compared with the poorly metastatic LNCaP indicating that up-regulation of TLR4 was positively correlated with metastasis of tumor cell. In the highly metastatic cancer cell PC3, gene silencing of TLR4 using siRNA significantly inhibited TLR4 mRNA expression and protein level. Knockdown of TLR4 in PC3 cells resulted in a dramatic reduction of tumor cell migration and invasion as indicated by a Matrigel invasion assay. Furthermore, TLR4 siRNA suppressed cell viability and ultimately caused the induction of apoptotic cell death. The effects were associated with abrogating TLR4-mediated signaling to downstream target molecules such as myeloid differentiation factor 88 (MyD88), adaptor-inducing IFN-beta (TRIF), and interferon regulatory factor-1 (IRF-1). In a mouse prostate cancer model, administration with the plasmid construct expressing siRNA for TLR4 obviously inhibited established tumor growth and survival. These studies revealed evidence of a multifaceted signaling network operating downstream of TLR4-mediated tumor cell invasion, proliferation, and survival. Thus, RNA interference-directed targeting of TLR4 may raise the potential of its application for cancer therapy.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Bmi-1 siRNA inhibited ovarian cancer cell line growth and decreased telomerase activity

The knockdown of Bmi-1 could effectively suppress cancer cell proliferation and tumourigenicity in several cancers. This study aims to investigate whether or not Bmi-1 plays a causative role in the proliferation of ovarian epithelial cancer cells and telomerase activity. The messenger RNA (mRNA) and protein expression levels of Bmi-1 in the human ovarian carcinoma cell line OVCAR-3 were downregulated by Bmi-1 siRNA, as confirmed by real-time polymerase chain reaction (PCR) and Western blot. Cell viability was analysed by MTT assay, and telomerase activity was analysed by a modified telomeric repeat amplification protocol. Targeting Bmi-1 with siRNA inhibited Bmi-1 mRNA over five-fold compared with the control cells, and inhibited Bmi-1 protein expression over three-fold compared with control cells. The viability of the OVCAR-3 ovarian cancer cell line was reduced by Bmi-1 mRNA compared to control cells. Telomerase activity was decreased 22.73% (from 0.33 to 0.255) by Bmi-1 siRNA treatment compared to control cells. As Bmi-1 siRNA depressed telomerase activity, cell immortalisation may be prevented; thus, silencing Bmi-1 may be a potential therapy to manage ovarian cancer.     

Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

Lymphatic endothelial cells actively regulate prostate cancer cell invasion

Lymphatic vessels serve as the primary route for metastatic spread to lymph nodes. However, it is not clear how interactions between cancer cells and lymphatic endothelial cells (LECs), especially within hypoxic microenvironments, affect the invasion of cancer cells. Here, using an MR compatible cell perfusion assay, we investigated the role of LEC–prostate cancer (PCa) cell interaction in the invasion and degradation of the extracellular matrix (ECM) by two human PCa cell lines, PC‐3 and DU‐145, under normoxia and hypoxia, and determined the metabolic changes that occurred under these conditions. We observed a significant increase in the invasion of ECM by invasive PC‐3 cells, but not poorly invasive DU‐145 cells when human dermal lymphatic microvascular endothelial cells (HMVEC‐dlys) were present. Enhanced degradation of ECM by PC‐3 cells in the presence of HMVEC‐dlys identified interactions between HMVEC‐dlys and PCa cells influencing cancer cell invasion. The enhanced ECM degradation was partly attributed to increased MMP‐9 enzymatic activity in PC‐3 cells when HMVEC‐dlys were in close proximity. Significantly higher uPAR and MMP‐9 expression levels observed in PC‐3 cells compared to DU‐145 cells may be one mechanism for increased invasion and degradation of matrigel by these cells irrespective of the presence of HMVEC‐dlys. Hypoxia significantly decreased invasion by PC‐3 cells, but this decrease was significantly attenuated when HMVEC‐dlys were present. Significantly higher phosphocholine was observed in invasive PC‐3 cells, while higher glycerophosphocholine was observed in DU‐145 cells. These metabolites were not altered in the presence of HMVEC‐dlys. Significantly increased lipid levels and lipid droplets were observed in PC‐3 and DU‐145 cells under hypoxia reflecting an adaptive survival response to oxidative stress. These results suggest that in vivo, invasive cells in or near lymphatic endothelial cells are likely to be more invasive and degrade the ECM to influence the metastatic cascade. Copyright © 2016 John Wiley & Sons, Ltd.     

Modulation of choline kinase activity in human cancer cells observed by dynamic 31P NMR

Choline metabolites are widely studied in cancer research as biomarkers of malignancy and as indicators of therapeutic response. However, endogenous phosphocholine levels are determined by a number of processes that confound the interpretation of these measurements, including membrane transport rates and a series of enzyme catalysed reactions in the Kennedy pathway. Employing a dynamic ³¹P NMR assay that is specific to choline kinase (ChoK) we have measured the rates of this enzyme reaction in cell lysates of MDA‐MB‐231 breast, PC‐3 prostate and HeLa cervical cancer cells and in solutions of purified human ChoK. The rates are sensitive to inhibition by hemicholinium‐3 (HC‐3), a competitive ChoK inhibitor, and to N‐[2‐bromocinnamyl(amino)ethyl]‐5‐isoquinolinesulphonamide (H‐89), an agent commercialized as a specific cyclic‐AMP‐dependent protein kinase A (PKA) inhibitor. Copyright © 2009 John Wiley & Sons, Ltd.