Tumor cells amino acid uptake

Ricin - A phytotoxic protein from Ricinus communis is inhibitory towards protein synthesis in experimental tumor cells. It has a moderate Inhibitory effect on DNA synthesis without affecting RNA synthesis. Its mode of action is not known, but is not due to impairment of glucose metabolism nor amino acid uptake in tumor cells.173... 

The in vitro release profiles of DOX from the polymeric micelles was studied in in PBS (0.1 M, pH 7.4) and acetate buffer solutions (0.1 M, pH 5.4) at 37 °C. The results showed an initial burst release of DOX and followed by a sustained release for about 48 h. The initial burst release of DOX from micelles could be attributed to the diffusion of DOX located close to the surface of particles or within the hydrophilic shell [6]. The total release of DOX in a period of 48 h with pH 7.4 and 5.4 was 25% and 37% of total DOX concentration, respectively. However, the release of DOX at a pH value of 5.4 was found faster than that at a pH value of 7.4. These results could be attributed to the re-protonation of the amino group of DOX and the faster degradation of the micelle core at lower pH values. This pH-dependent release profile is of particular interest. It is expected that the greater part of DOX-loaded micelles will remain in the micelles cores for a considerable time period in plasma after intravenous administration and have the potential for prolonged DOX retention time in the blood circulatioa However, a faster release may occur at low local pH surrounding the tumor site or by the more acidic environment inside the endosome and lysosome of tumor cells after cellular uptake of micelles through endocytosis. 

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

The molecular targeting agent (C) selected from a representative sequence of amino acids, resulting in uptake of the triade system particular in the tumor cells. 

Carcinoids are the most common tumors arising from the diffuse neuroendocrine system of the GI tract and pancreas. Derived primarily from enterochromaffin cells, these tumors are widely distributed in the body but found with greatest frequency in the GI tract (74%) and respiratory tract (25%). Carcinoids are often classified as APUDomas (flmine precursor uptake and decarboxylation) because of the ability of enterochromaffin cells to take up and decarboxylate amino acid precursors of biogenic amines. In this regard, carcinoid tumors share certain pathological and biological similarities with pheochromocytomas. 

All animal cells studied up to now show the abiUty to accumulate certain amino acids against their concentration gradients, while for sugars such accumulation was only observed in polarized cells, e.g. cells from small intestine or proximal tubule. Ehrlich cells and other tumor cell lines [53-57], tissue culture cells, avian erythrocytes [58], isolated cells and slices from different tissues [59] were studied for their ability to accumulate amino acids. Apart from investigations with epithelia, studies, on Ehrlich cells, of the energetics and mechanism of the accumulative uptake of amino acids play a fundamental role in working out the concept of cotransport between Na and amino acids by animal cells. 

C-labeled methionine (MET), -labeled alpha-meth-yltyrosine (IMT), and F-labeled 0-(2-fluoroethyl)-L-tyrosine (FET) are the most important radiolabeled amino acids used in the diagnosis of brain tumors. These three tracers have shown a very similar uptake intensity and distribution in brain tumors (Langen et al. 1997 Weber et al. 2000). Currently available amino acid-PET tracers are accumulated by L and A amino acid transporters. Tumor cells take up radiolabeled amino acids at a high rate, while there is only a relatively low uptake in normal cerebral tissue. At the level of the blood-brain barrier (BBB) they are independent from the BBB disturbance. 

The majority of PET studies in oncology have been performed with C-labeled methionine, which is relatively easy to synthesize (41-43). Because of its low uptake in the brain, ["Cjmethionine has been proven to be a sensitive and specific tool for brain tumors, able to differentiate nonneoplastic from malignant lesions (42,44). Methionine uptake reflects the amino acid transport, whereas ["Cjtyrosine has been used to quantify protein synthesis rate in brain tumors (45,46). An I-iodinated tyrosine analog (IMT) also has been proposed to assess tumor cells proliferation by means of SPECT imaging. IMT uptake in glioma cells in vitro (47) and in soft-tissue sarcomas in vivo (48) was shown to be induced by rapid proliferation. 

Because cell growth requires an adequate energy supply, FDG uptake has often been used as an indirect measure of tumor proliferation (57). Correlation between FDG uptake and tumor cells proliferation was confirmed in ghoma (58), meningioma (59), lymphoma (60), ovarian epithelial carcinoma (57), lung cancer (61-63), and squamous-cell carcinoma of the head and neck (64). 1-[ C]Acetate, ["C]choline, and radiolabeled amino acids are other PET tracers used to indirectly measure tumor proliferation, because cell growth and proliferation inevitably necessitate membrane constiments and proteins (65). 

The main factor for the increased uptake of radiolabeled amino acids in malignant tumors appears to be the increased expression and activity of the so-called L-type amino acid transporter. This transport system mediates the movement of neutral amino acids across the cell membrane. It has long been characterized as a nonenergy-dependent bidirectional transport system, which... 

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