Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells

Abstract

Summary We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31 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 intemal 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 < 0.006) and phosphocreatine (PCr) (P < 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.

Colorectal Tumor Cells Treated with 5-FU, Oxaliplatin, Irinotecan, and Cetuximab Exhibit Changes in18F-FDG Incorporation Corresponding to Hexokinase Activity and Glucose Transport

“…Thus, NTP levels in the colonic tumor cell line HT-29 cells treated with nitrogen mustard (29) and in spheroids of MCF-7 and T47D breast tumor cells treated with doxorubicin during a 24-h period (30) increased, but treatment of SW620 cells with doxorubicin did not cause an increase in ATP content (31). However, in our study irinotecan treatment was associated with increased ATP levels in SW620 cells after a 48-h and also in HCT-8 cells after a 72-h treatment with irinotecan.…”

“…Several studies using 31 P-nuclear magnetic resonance spectroscopy have shown that nucleotide triphosphate (NTP) (mainly ATP) levels can increase in cells responding to treatment (29)(30)(31). Thus, NTP levels in the colonic tumor cell line HT-29 cells treated with nitrogen mustard (29) and in spheroids of MCF-7 and T47D breast tumor cells treated with doxorubicin during a 24-h period (30) increased, but treatment of SW620 cells with doxorubicin did not cause an increase in ATP content (31).…”

Metabolic effects of photodynamically induced apoptosis in an erythroleukemic cell line. A31P NMR spectroscopic study of Victoria-Blue-BO-sensitized TF-1 cells

“…Over the past years, several reports have clearly established that apoptosis is an energy-dependent process in its early stages and therefore requires intact mitochondria and ATP content. Furthermore, ATP elevation preceding apoptosis has been reported by Sanchez-Alcazar et al (1997) for TNF-␣ combination therapies of metabolically inactivated L929 cells, and by Boddie et al (1998) for nitrogen-mustard treatment of HT29 cells. Interestingly, a transient increase in NTP content before apoptosis has also been reported in the case of photodynamically treated BHK or MH22 cells, with Temoporfin used as sensitizer (Kirveliene et al, 1997).…”

“…We used in vitro 31 P NMR spectroscopy of wholecell suspensions to analyze cellular metabolism at various time points during the first 8 hr following PDT treatment, and employed DNA electrophoresis to test the hypothesis of programmed cell death. Other authors have shown that apoptotic cell death was accompanied by reduced PtdCho synthesis in neutrophils (Nunn et al, 1996) and in some cases by either a decrease or an increase in CDP-choline content (Boggs et al, 1998;Williams et al, 1998), suggesting strong perturbations in phospholipid metabolism and in the phosphatidylcholine pathway in particular. Moreover, a link has been established between perturbations of the phosphatidylcholine pathway and the generation of ceramide (Wieder et al, 1998), a potent inducer of apoptosis (Hannun and Obeid, 1995).…”

Aspirin induces cell death and caspase-dependent phosphatidylserine externalization in HT-29 human colon adenocarcinoma cells

“…On the other hand, nitrogen mustard induces apoptosis of HT-29 cells in the absence of DNA laddering (Boddie et al, 1998).…”