Localized Hypoxia Results in Spatially Heterogeneous Metabolic Signatures in Breast Tumor Models

Tumor hypoxia triggers signaling cascades that significantly affect biologic outcomes such as resistance to radiotherapy and chemotherapy in breast cancer. Hypoxic regions in solid tumor are spatially heterogeneous. Therefore, delineating the origin and extent of hypoxia in tumors is critical. In this study, we have investigated the effect of hypoxia on different metabolic pathways, such as lipid and choline metabolism, in a human breast cancer model. Human MDA-MB-231 breast cancer cells and tumors, which were genetically engineered to express red fluorescent tdTomato protein under hypoxic conditions, were used to investigate hypoxia. Our data were obtained with a novel three-dimensional multimodal molecular imaging platform that combines magnetic resonance (MR) imaging, MR spectroscopic imaging (MRSI), and optical imaging of hypoxia and necrosis. A higher concentration of noninvasively detected total choline-containing metabolites (tCho) and lipid CH3 localized in the tdTomato-fluorescing hypoxic regions indicated that hypoxia can upregulate tCho and lipid CH3 levels in this breast tumor model. The increase in tCho under hypoxia was primarily due to elevated phosphocholine levels as shown by in vitro MR spectroscopy. Elevated lipid CH3 levels detected under hypoxia were caused by an increase in mobile MR-detectable lipid droplets, as demonstrated by Nile Red staining. Our findings demonstrate that noninvasive MRSI can help delineate hypoxic regions in solid tumors by means of detecting the metabolic outcome of tumor hypoxia, which is characterized by elevated tCho and lipid CH3.     

NO-sulindac inhibits the hypoxia response of PC-3 prostate cancer cells via the Akt signalling pathway

Nitric oxide-donating non-steroidal anti-inflammatory drugs are safer than traditional NSAIDs and inhibit the growth of prostate cancer cells with greater potency than NSAIDs. In vivo, prostate cancer deposits are found in a hypoxic environment which induces resistance to chemotherapy. The aim of this study was to assess the effects and mechanism of action of a NO-NSAID called NO-sulindac on the PC-3 prostate cancer cell line under hypoxic conditions. NO-sulindac was found to have pro-apoptotic, cytotoxic, and anti-invasive effect on PC-3 cells under normoxia and hypoxia. NO-sulindac was significantly more cytotoxic than sulindac at all oxygen levels. The sulindac/linker and NO-releasing subunits both contributed to the cytotoxic effects of NO-sulindac. Resistance of PC-3 cells to NO-sulindac was induced as the oxygen concentration declined. Hypoxia-induced chemoresistance was reversed by knocking-down hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA using RNAi. Nuclear HIF-1alpha levels were upregulated at 0.2% oxygen but reduced by treatment with NO-sulindac, as was Akt phosphorylation. NO-sulindac treatment of hypoxic PC-3 cells transfected with a reporter construct, downregulated activation of the hypoxia response element (HRE) promoter. Co-transfection of PC-3 cells with the HRE promoter reporter construct and myr-Akt (constitutively active Akt) plasmids reversed the NO-sulindac induced reduction in HRE activation. Real-time polymerase chain reaction analysis of hypoxic, NO-sulindac treated PC-3 cells showed downregulation of lysyl oxidase and carbonic anhydrase IX mRNA expression. Collectively, these novel findings demonstrate that NO-sulindac directly inhibits the hypoxia response of PC-3 prostate cancer cells by inhibiting HIF-1alpha translation via the Akt signalling pathway. The ability of NO-sulindac to inhibit tumour adaption to hypoxia has considerable relevance to the future management of prostate cancer with the same cellular properties as PC-3.     

Multimodal mass spectrometric imaging of small molecules reveals distinct spatio-molecular signatures in differentially metastatic breast tumor models

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.     

Choline kinase down-regulation increases the effect of 5-fluorouracil in breast cancer cells

Identifying strategies to increase cancer cell kill while sparing normal tissue is critically important in cancer chemotherapy. Choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC), is elevated in cancer cells and presents a novel target for increasing cell kill. Here, we have examined the effects of transiently down-regulating Chk by small interfering RNA against Chk (siRNA-chk) on PC and total choline-containing compound (tCho) levels and on the viability/proliferation of estrogen receptor-negative and estrogen receptor-positive breast cancer cell lines and a nonmalignant mammary epithelial cell line. We investigated the effects of combination treatment with transient siRNA-chk transfection and the anticancer drug 5-fluorouracil (5-FU) in those cell lines. Microarray analysis of the invasive estrogen receptor-negative MDA-MB-231 cell line was done to characterize molecular changes associated with Chk down-regulation. Chk down-regulation decreased PC and tCho levels in the malignant cell lines, whereas the cell viability/proliferation assays detected a decrease in proliferation in these cells. In contrast, Chk down-regulation had an almost negligible effect on PC and tCho levels as well as cell viability/proliferation in the nonmalignant cell line. A combination of siRNA-chk with 5-FU treatment resulted in a larger reduction of cell viability/proliferation in the breast cancer cell lines; this reduction was evident to a much lesser degree in the nonmalignant cells. Microarray analysis showed that Chk down-regulation affected 33 proliferation-related genes and 9 DNA repair-related genes. Chk down-regulation with siRNA-chk may provide a novel alternative to enhance the effect of anticancer drugs in malignant cells.     

Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha)

Hypoxia-inducible factor-1alpha (HIF-1alpha) is both a potential endogenous marker of tumor hypoxia and therapeutic target, and elevated HIF-1alpha protein levels have been shown to be associated with increased hypoxic radiation resistance in FaDu human pharyngeal carcinoma cells in vitro. Here, we found that in FaDu xenografts, no significant HIF-1alpha protein accumulation was detectable by either flow cytometry or Western blot, despite the presence of hypoxic (pimonidazole-positive, radiation resistant) cells. To investigate the effect of different tumor microenvironment conditions on hypoxic HIF-1alpha accumulation, we performed in vitro hypoxia experiments (0.1% O2, 24 h) with manipulation of pH (7.4 vs. 6.7), glucose (0-5.5 mM) and serum (0 or 10%) availability in FaDu and HT 1080 human fibrosarcoma cells. Hypoxic induction of HIF-1alpha protein was strongly dependent on glucose availability and largely abolished at 0.55 mM glucose or less in both cell lines. This glucose effect was confirmed in a hypoxia-responsive-element (HRE)/enhanced-green-fluorescent-protein (EGFP) reporter assay in transfected HT 1080 cells and possibly explains a lack of HIF-1alpha protein in hypoxic tumor cells.     

PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells

Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer.     

缺氧条件下YC-1对人胰腺癌细胞VEGF和GPI基因的抑制效应

目的探讨缺氧条件下3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole(YC-1)对人胰腺癌细胞血管内皮生长因子(VEGF)和磷酸葡萄糖异构酶(GPI)基因的调控作用及其机制。方法缺氧条件下体外培养胰腺癌PC-3细胞株,免疫细胞化学染色法检测常氧和缺氧条件下缺氧诱导因子- 1α(HIF-1α)的表达。半定量RT-PCR检测YC-1对PC-3细胞VEGF、GPI、HIF-1αmRNA表达的调节作用。Western blot检测YC-1对PC-3细胞HIF-1α蛋白表达的调节作用。MTT法检测YC-1对缺氧PC-3细胞恶性增殖的影响。结果缺氧条件下,HIF-1α主要表达于PC-3细胞胞核内。随着YC-1浓度升高,VEGF和GPI mRNA表达、HIF-1α蛋白表达逐渐受抑,并呈明显的剂量依赖性;实验组HIF- 1αmRNA均呈强表达,其表达水平不随YC-1作用浓度的升高而降低。缺氧条件下,实验组细胞增殖抑制率均增高,100μmol／L YC-1组细胞增殖抑制率达73．28%±2．01％。结论缺氧状态下,YC-1抑制人胰腺癌PC-3细胞VEGF和GPI基因转录与其抑制HIF-1α蛋白的表达有关,YC-1能够抑制PC-3细胞生长和增殖。     

Metabolic signatures imaged in cancer-induced cachexia

Cancer-induced cachexia is a complex and poorly understood life-threatening syndrome that is characterized by progressive weight loss due to metabolic alterations, depletion of lipid stores, and severe loss of skeletal muscle protein. Gaining the ability to noninvasively image the presence or onset of cachexia is important to better treat this condition, to improve the design and optimization of therapeutic strategies, and to detect the responses to such treatments. In this study, we employed noninvasive magnetic resonance spectroscopic imaging (MRSI) and [(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) positron emission tomography (PET) to identify metabolic signatures typical of cachectic tumors, using this information to analyze the types and extents of metabolic changes induced by the onset of cachexia in normal tissues. Cachexia was confirmed by weight loss as well as analyses of muscle tissue and serum. In vivo, cachexia-inducing murine adenocarcinoma (MAC)16 tumors were characterized by higher total choline (tCho) and higher (18)FDG uptake than histologically similar noncachectic MAC13 tumors. A profound depletion of the lipid signal was observed in normal tissue of MAC16 tumor-bearing mice but not within the tumor tissue itself. High-resolution (1)H magnetic resonance spectroscopy (MRS) confirmed the high tCho level observed in cachectic tumors that occurred because of an increase of free choline and phosphocholine. Higher succinate and lower creatine levels were also detected in cachectic tumors. Taken together, these findings enhance our understanding of the effect of cancer on host organs and tissues as well as promote the development of noninvasive biomarkers for the presence of cachexia and identification of new therapeutic targets.     

Ibuprofen-mediated reduction of hypoxia-inducible factors HIF-1alpha and HIF-2alpha in prostate cancer cells.

PURPOSE: Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are considered to be potential targets for antineoplastic therapy because they regulate the expression of genes that contribute to tumor cell survival, aggressiveness, and angiogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) have gained considerable interest as anticancer agents because of their cytotoxic and antiangiogenic properties. The aim of this study was to investigate whether NSAIDs inhibit HIFs and HIF-regulated gene expression in prostate cancer cells. Experimental Design: PC3 and DU-145 cells were treated with ibuprofen (Ibu) and other NSAIDs under normoxic and hypoxic (95% N(2), 5% CO(2); <10 ppm O(2)) conditions. The effect of NSAIDs on HIF proteins was analyzed by Western blot analysis. HIF-regulated proteins, vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1), were analyzed by ELISA and Western blot analysis, respectively. RESULTS: Exposure of PC3 and DU-145 cells to hypoxic condition up-regulated HIF-1alpha and HIF-2alpha proteins. Treatment with Ibu under normoxic and hypoxic conditions reduced the level of HIF-1alpha and HIF-2alpha. Ibu-mediated down-regulation of HIFs was associated with down-regulation of HIF-regulated proteins VEGF and Glut-1 in cells exposed to hypoxia. Other nonspecific NSAIDs, diclofenac and ketorolac, also inhibited HIF-1alpha and HIF-2alpha. The reduction in HIFs was observed in PC3 cells that expressed cyclooxygenase-2 (COX-2) protein as well as in DU-145 cells, which did not express COX-2 protein. COX-2-specific inhibitor NS-398 did not inhibit HIF-1alpha or VEGF and GLUT-1. CONCLUSIONS: These data indicate that one of the effects of NSAIDs is to reduce HIF protein levels. The inhibition of HIFs by NSAIDs was COX-2 independent.     

Resistance to caspase-dependent, hypoxia-induced apoptosis is not hypoxia-inducible factor-1 alpha mediated in prostate carcinoma cells

BACKGROUND: Hypoxia occurs in association with cancer development, the result being a more aggressive and metastatic cancer phenotype. Hypoxia, which activates hypoxia-inducible factor-1 alpha (HIF-1alpha), is associated with a number of cellular changes including increased apoptotic resistance. The authors hypothesized that HIF-1alpha is central to the cell's ability to resist apoptosis induced during the hypoxia selection process. METHODS: PWR-1E, LNCaP, LNCaP-HOF, PC-3, and DU-145 cells were cultured in normoxic and hypoxic conditions. Apoptosis was assessed by propidium iodide DNA staining. Cleavage of specific substrates was used to assess caspase activity and Western blotting was used to assess mitochondrial release of cytochrome c and second mitochondria-derived activator caspase (SMAC)/Diablo. A dominant negative HIF-1alpha construct was transfected into the PC-3 and LNCaP cells to block HIF-1alpha activity. RESULTS: PC-3 and DU-145 were resistant to apoptosis induced by exposure to hypoxia, but the PWR-1E and LNCaP cells were susceptible. This induction of apoptosis in the LNCaP cells was caspase dependent but independent of cytochrome c release. Blocking the activity of HIF-1alpha had no effect on increased apoptotic susceptibility in the PC-3 cells. LNCaP-HOF cells, which were resistant to hypoxia-induced apoptosis, showed no increase in HIF-1alpha expression or activity. CONCLUSIONS: Apoptotic resistance is already established in cells that survive a hypoxic insult and whereas increased HIF-1alpha activity may be essential for the development of a more aggressive cancer phenotype, it may not be responsible for the initial selection of an apoptotic resistance phenotype.