Abnormal choline phospholipid metabolism is a hallmark of cancer. The magnetic resonance spectroscopy (MRS) detected total choline (tCho) signal can serve as an early noninvasive imaging biomarker of ...chemotherapy response in breast cancer. We have quantified the individual components of the tCho signal, glycerophosphocholine (GPC), phosphocholine (PC) and free choline (Cho), before and after treatment with the commonly used chemotherapeutic drug doxorubicin in weakly metastatic human MCF7 and triple-negative human MDA-MB-231 breast cancer cells. While the tCho concentration did not change following doxorubicin treatment, GPC significantly increased and PC decreased. Of the two phosphatidylcholine-specific PLD enzymes, only PLD1, but not PLD2, mRNA was down-regulated by doxorubicin treatment. For the two reported genes encoding GPC phosphodiesterase, the mRNA of GDPD6, but not GDPD5, decreased following doxorubicin treatment. mRNA levels of choline kinase α (ChKα), which converts Cho to PC, were reduced following doxorubicin treatment. PLD1 and ChKα protein levels decreased following doxorubicin treatment in a concentration dependent manner. Treatment with the PLD1 specific inhibitor VU0155069 sensitized MCF7 and MDA-MB-231 breast cancer cells to doxorubicin-induced cytotoxicity. Low concentrations of 100 nM of doxorubicin increased MDA-MB-231 cell migration. GDPD6, but not PLD1 or ChKα, silencing by siRNA abolished doxorubicin-induced breast cancer cell migration. Doxorubicin induced GPC increase and PC decrease are caused by reductions in PLD1, GDPD6, and ChKα mRNA and protein expression. We have shown that silencing or inhibiting these genes/proteins can promote drug effectiveness and reduce adverse drug effects. Our findings emphasize the importance of detecting PC and GPC individually.
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.
Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging ...(MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimonidazole as an exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimonidazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides, and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly colocalized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.
Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of ...orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes.
: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively.
: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively.
: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.
The integration of advances in molecular biology, synthetic chemistry and visualization techniques has catapulted imaging into a molecular–functional realm, so that imaging is finding basic-research, ...preclinical and translational applications in cancer. Currently, molecular-imaging capabilities include the ability to image gene expression, receptors, signaling pathways, apoptosis, multidrug resistance and the extracellular matrix (ECM). Functional-imaging capabilities include the ability to assess angiogenesis, hypoxia and metabolism. Traditionally, imaging has played an important role in cancer diagnosing and determining response to treatment. However, it is the realization of the goal of noninvasively visualizing molecules and molecular pathways and relating these to function that makes multi-modality imaging such an exciting and powerful means for studying a multifaceted disease such as cancer.
Over the past decade, our program has focused on understanding the role of the physiological environment, tumor vasculature, and metabolism in several of the aggressive phenotypic traits of cancer, ...such as invasion and metastasis. These studies have been performed primarily with magnetic resonance (MR) imaging (MRI) and spectroscopy (MRS) on human breast and prostate cancer models. During the course of these studies, we observed specific changes in choline phospholipid metabolism associated with a more aggressive phenotype. Molecular or pharmacologic interventions that reduced this aggressiveness were also consistent with a reversal of these alterations. In this contextual review, we have outlined the insight we have gained from these studies and have discussed some of the enzymes and pathways that may present novel targets for pharmaceutical interventions in cancer. Keywords: Choline phospholipid metabolism; cancer; magnetic resonance spectroscopy
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