phosphocreatine and Colonic-Neoplasms

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.

Topics: Adenosine Triphosphate; Ammonia; Antineoplastic Agents, Alkylating; Apoptosis; Cell Cycle; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cytidine Triphosphate; Genes, bcl-2; Glutathione; Guanosine Triphosphate; HT29 Cells; Humans; Magnetic Resonance Spectroscopy; Mechlorethamine; Phosphocreatine; Purine Nucleotides; Up-Regulation; Uridine Triphosphate

The brain isoform of creatine kinase has been implicated in cellular transformation processes. Cyclocreatine, a creatine kinase substrate analog, was previously shown to be cytotoxic to a broad spectrum of solid tumors. We have synthesized, enzymatically characterized, and evaluated the antitumor activity of a series of substrate analogs of creatine kinase. Using in vitro assays, we demonstrate that several of these analogs are cytotoxic to the human ME-180 cervical carcinoma, the MCF-7 breast adenocarcinoma and the HT-29 colon adenocarcinoma cell lines at low mM concentrations. Analogs that were active in vitro delayed the growth of a subcutaneously implanted rat 13,762 mammary adenocarcinoma. Tumor growth delays of 6-8 days were achieved, which is comparable to effects seen with standard regimens of currently used anticancer drugs. These studies further establish the creatine kinase system as a promising and novel target for anticancer chemotherapy drug design.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Death; Colonic Neoplasms; Creatine; Creatine Kinase; Creatinine; Female; Humans; Neoplasms, Experimental; Phosphocreatine; Rats; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Flavone acetic acid and 5,6-dimethyl xanthenone acetic acid have a broad spectrum of anti-tumor activity in mice, and act by stimulating immune cells and inhibiting tumor blood flow, resulting in hemorrhagic necrosis within 24 hrs. To study the evolution of hemorrhagic necrosis, subcutaneous Colon 38 tumors were examined by light and electron microscopy from 30 min to 24 hrs after treatment with these agents, and measurements of tumor energy metabolites made. The results show that both agents cause apoptosis beginning at 30 min, and that by 4 hrs necrosis supervenes, accompanied by rupture of tumor blood vessels. The absence of early endothelial cell damage or thrombosis suggests that vessel rupture, and consequent loss of blood flow and energy metabolite depletion, is caused by loss of extravascular mechanical support by the tumor parenchyma.

Topics: Adenocarcinoma; Adenosine Triphosphate; Animals; Apoptosis; Colonic Neoplasms; Flavonoids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Microscopy, Electron; Necrosis; Phosphocreatine; Xanthenes; Xanthones

Rhodamine 123 is a lipophilic cationic compound that is selectively taken up by cancer cell mitochondria. This compound is toxic to epithelial cancer cells in vitro and displays significant anticancer activity in vivo. However, the mechanism of action of rhodamine 123 in intact, actively metabolizing cell preparations is unknown. We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to quantitatively characterize how rhodamine 123 affects the energetics of human colon cancer cells (HCT-116) and spontaneously immortalized normal epithelial cells (CV-1). Rhodamine 123 differentially altered the phosphorus and glucose metabolism of HCT-116 and CV-1 cells. 31P-nuclear magnetic resonance detected mitochondrial poisoning in the HCT-116 human colon cancer cell line in its early stages after selective uptake of rhodamine 123. When we compared administration of rhodamine 123 and [1-C13]glucose to administration of [1-C13]glucose alone in the HCT-116 cells, we noted a marked decrease in intracellular pH to 6.7 +/- 0.06 (mean +/- SD) units, a 2.2-fold increase in lactate production, and a 1.8-fold increase in glucose consumption after 10 h. In addition, we found a 2-fold rise in intracellular free magnesium 12 h after rhodamine 123 administration. These results suggest that when rhodamine 123 inhibits mitochondrial ATP production, it initially stimulates cytoplasmic glycolysis in an attempt to maintain cellular energy demands. The marked fall in intracellular pH and rise in intracellular free magnesium after administration of rhodamine 123 may inhibit activity of several glycolytic enzymes: this effect would inhibit cytoplasmic ATP generation and interfere with multiple cell enzymatic processes, leading to cell death. The CV-1 cells showed no change in intracellular pH, intracellular free magnesium, or magnesium-bound ATP levels over the 24-h period following rhodamine 123 administration. Rhodamine 123 also failed to alter glucose utilization and lactate production levels significantly in the CV-1 cells. These results prove the usefulness of 31P- and 13C-nuclear magnetic resonance spectroscopy for quantifying differing effects of rhodamine 123 on the high energy phosphate metabolism and glucose metabolism of HCT-116 and CV-1 cells.

Topics: Adenosine Triphosphate; Antimetabolites, Antineoplastic; Carbon; Colonic Neoplasms; Humans; Hydrogen-Ion Concentration; Magnesium; Magnetic Resonance Spectroscopy; Phosphocreatine; Rhodamine 123; Rhodamines; Tumor Cells, Cultured

Nuclear magnetic resonance spectroscopy is increasingly used to study cellular metabolism in a manner respecting cell integrity. The contribution of phosphorus-31 and carbon-13 NMR is discussed and illustrated by specific examples taken from work carried out in this laboratory or from the literature. A particular emphasis is layed on metabolite identification, quantitation and fluxes as studied by phosphorus-31 NMR which provides a direct insight of energy metabolism. The analysis of perfused rat liver by natural abundance carbon-13 NMR illustrates the potential of the method of study non-invasively lipid and carbohydrate metabolism in living systems. The use of carbon-13 enriched substrates to pinpoint a specific pathway of the intermediary metabolism is described in the case of excised rat heart and liver perfused with (2-C13)-acetate. Clinical applications of NMR spectroscopy are rapidly reviewed.

Topics: Adenine Nucleotides; Adenocarcinoma; Animals; Carbon; Cell Line; Cells; Colonic Neoplasms; Humans; Liver; Magnetic Resonance Spectroscopy; Myocardium; Phosphocreatine; Phosphorus; Rats; Spectrum Analysis

Topics: Adenosine Triphosphate; Animals; Breast Neoplasms; Cell Line; Colonic Neoplasms; Female; Humans; Hydrogen-Ion Concentration; Lung Neoplasms; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Phosphates; Phosphocreatine; Transplantation, Heterologous