Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Effect of selenium on the growth of three human colon cancer cell lines

The effects of selenium were investigated on three human colon cancer cell lines: Caco 2, HRT 18, and HT 29. At low concentrations (10-100 nM), selenium stimulated cell growth in serum-free medium. Thus, selenium is an essential trace element for cell proliferation. At higher concentrations, selenium inhibited cell growth. The rate of 75Se uptake was the same in all of the cell lines studied, but the quantity incorporated differed. GSH-Px activity was dependent on the selenium content of the medium. DNA and protein synthesis paralleled the growth curve. Comparison with the curve of viability revealed that selenium inhibited cell growth in two ways: by inhibiting DNA synthesis, without affecting cell viability, and, at higher doses, by cytotoxicity.     

Apoptosis induced by selenium in human glioma cell lines

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced the typical ladder pattern of DNA fragmentation commonly found in apoptosis), which were prevented by catalase. Few effects of selenium on NTI4 fibroblasts were found. These findings demonstrate that selenium may induce, by apoptosis, cell death of human glioma cell lines, which are resulting from free radical oxygen forming.     

Iron metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH₄Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (⁵⁵Fe/⁵⁹Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.     

Differential metabolism of mannose by Chinese hamster cell lines

In contrast with the CHO-Kl Chinese hamster cell line, the V79 cell line was found not to grow on the sugar mannose, and in fact to be inhibited and killed. Study showed no differences between the lines with regard to mannose transport, mannose specific energy metabolism, or effect of mannose on energy or nucleic acid metabolism. The V79 line lacked an α-mannosidase which the CHO-Kl line possessed.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Hypoxia and cancer cell metabolism

The past decade has witnessed a rapid accumulation of evi- dence showing that hypoxic microenvironment, which is typical during cancer development, plays key roles in regu- lating cancer cell metabolism. In this review, we will focus on the role of hypoxic response, particularly, its master regulator hypoxia-inducible factor-I, in regulating glucose, lipid, as well as amino acid metabolism in cancer cells. We will also discuss the therapeutic opportunities by targeting specific pathways that facilitate metabolic reprogramming in cancer cells.     

Autophagy and cancer cell metabolism

Autophagy is a catabolic process involving lysosomal turnover of proteins and organelles for maintenance of cellular homeostasis and mitigation of metabolic stress. Autophagy defects are linked to diseases, such as liver failure, neurodegeneration, inflammatory bowel disease, aging and cancer. The role of autophagy in tumorigenesis is complex and likely context-dependent. Human breast, ovarian and prostate cancers have allelic deletions of the essential autophagy regulator BECN1 and Becn1(+/-) and other autophagy-deficient transgenic mice are tumor-prone, whereas tumors with constitutive Ras activation, including human pancreatic cancers, upregulate basal autophagy and are commonly addicted to this pathway for survival and growth; furthermore, autophagy suppression by Fip200 deletion compromises PyMT-induced mammary tumorigenesis. The double-edged sword function of autophagy in cancer has been attributed to both cell- and non-cell-autonomous mechanisms, as autophagy defects promote cancer progression in association with oxidative and ER stress, DNA damage accumulation, genomic instability and persistence of inflammation, while functional autophagy enables cancer cell survival under stress and likely contributes to treatment resistance. In this review, we will focus on the intimate link between autophagy and cancer cell metabolism, a topic of growing interest in recent years, which has been recognized as highly clinically relevant and has become the focus of intense investigation in translational cancer research. Many tumor-associated conditions, including intermittent oxygen and nutrient deprivation, oxidative stress, fast growth and cell death suppression, modulate, in parallel and in interconnected ways, both cellular metabolism and autophagy to enable cancer cells to rapidly adapt to environmental stressors, maintain uncontrolled proliferation and evade the toxic effects of radiation and/or chemotherapy. Elucidating the interplay between autophagy and tumor cell metabolism will provide unique opportunities to identify new therapeutic targets and develop synthetically lethal treatment strategies that preferentially target cancer cells, while sparing normal tissues.     

Metabolic signatures in apoptotic human cancer cell lines

Cancer cells have several specific metabolic features, which have been explored for targeted therapies. Agents that promote apoptosis in tumors are currently considered as a powerful tool for cancer therapeutics. The present study aimed to design a fast, reliable and robust system for metabolite measurements in cells lines to observe impact of apoptosis on the metabolome. For that purpose the NBS (newborn screen) mass spectrometry-based metabolomics assay was adapted for cell culture approach. In HEK 293 and in cancer cell lines HepG2, PC3, and MCF7 we searched for metabolic biomarkers of apoptosis differing from that of necrosis. Already nontreated cell lines revealed distinct concentrations of metabolites. Several metabolites indicative for apoptotic processes in cell culture including aspartate, glutamate, methionine, alanine, glycine, propionyl carnitine (C3-carnitine), and malonyl carnitine (C3DC-carnitine) were observed. In some cell lines metabolite changes were visible as early as 4?h after apoptosis induction and preceeding the detection by caspase 3/7 assay. We demonstrated for the first time that the metabolomic signatures might be used in the tests of efficacy of agents causing apoptosis in cell culture. These signatures could be obtained in fast high-throughput screening.     

S-adenosylhomocysteine metabolism in different cell lines: effect of hypoxia and cell density.

BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.     

Clinical aspects of selenium metabolism

Whereas defined selenium deficiency diseases are well characterized in animals, analogous syndromes in humans are unknown or occur only rarely under conditions of extreme selenium depletion. However, selenium deficiency has since been recognized to play important secondary roles in a variety of human diseases, and several of these can be prevented or treated by means of selenium supplementation. In clinical practice, the selenium status of patients should be monitored routinely, and corrective measures should be instituted to replete the Se body stores of patients where necessary. Interactions of Se with other elements are also of potentially great clinical significance. The importance of such interactions is exemplified by the effects of cadmium on selenium metabolism in spontaneously hypertensive rats. Deceased.     

Cell cycle regulators in cancer cell metabolism

Cancer proliferation and progression involves altered metabolic pathways as a result of continuous demand for energy and nutrients. In the last years, cell cycle regulators have been involved in the control of metabolic processes, such as glucose and insulin pathways and lipid synthesis, in addition to their canonical function controlling cell cycle progression. Here we describe recent data demonstrating the role of cell cycle regulators in the metabolic control especially in studies performed in cancer models. Moreover, we discuss the importance of these findings in the context of current cancer therapies to provide an overview of the relevance of targeting metabolism using inhibitors of the cell cycle regulation.     

Post-transcriptional regulation of P-glycoprotein expression in cancer cell lines

The present study of inhibitors shows that the histone deacetylase-induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5' end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5' untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA.     

Differential metabolism of guanine nucleosides by human lymphoid cell lines

Deficiency of the enzyme purine nucleoside phosphorylase is associated with a specific depletion of T cells which is presumably mediated by its substrate, 2'-deoxyguanosine. Inhibitors of this enzyme are therefore being developed as potential immunosuppressive agents. We have compared the effects of 8-aminoguanosine, a competitive inhibitor of purine nucleoside phosphorylase, on the metabolism of 2'-deoxyguanosine by human T lymphoblasts, B lymphoblasts, and mature T-cell lines. 8-Aminoguanosine markedly potentiates the accumulation of dGTP in T lymphoblasts, but results in increased GTP levels in B lymphoblasts and mature T cells. GTP accumulation is associated with ATP depletion of a magnitude similar to that seen with an inhibitor of de novo purine biosynthesis, but does not result in inhibition of either DNA or RNA synthesis. In contrast, direct inhibition of de novo purine biosynthesis sharply decreased the incorporation of [3H]uridine into both DNA and RNA. We conclude that the mechanism of cell damage resulting from prolonged accumulation of GTP appears to involve more than inhibition of de novo purine biosynthesis and consequent ATP depletion. Perturbations in guanine nucleotide pools resulting from partial inhibition of purine nucleoside phosphorylase activity in vivo could result in cellular toxicity not limited to the target T cell population.